Compositions and methods for treating eye infections and disease

ABSTRACT

The present invention provides compositions and methods for identifying subjects suffering from dry eye that can be treated by topical administration of a composition comprising lacritin or a bioactive fragment thereof. The application discloses in part that a ˜90 KDa deglycanated form of syndecan-1 is abundant in tears of normal individuals but not individuals suffering from dry eye, whereas a ˜25 kDa syndecan-1 fragment is detectable in dry, but not normal tears.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/125,357, filed Sep. 12, 2016, which is a U.S. national counterpartapplication of PCT International Application Serial No.PCT/US2015/019964, filed Mar. 11, 2015, which claims priority to U.S.Provisional Application Ser. No. 62/019,476, filed Jul. 1, 2014, andU.S. Provisional Application Ser. No. 61/951,680, filed Mar. 12, 2014,the disclosures of all which are hereby incorporated herein by referencein their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.EY013143 and EY018222, awarded by The National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 19 kilobytes ACII (Text) file named“266368SeqListing.txt,” created on Jun. 1, 2017.

BACKGROUND

Health of the ocular surface is dependent on tear fluid secretions fromthe lacrimal gland. The lacrimal acinar cells comprising the lacrimalgland are polarized and highly differentiated tear secreting cells thatadhere to a complex periacinar basement membrane. The bulk of the apicalcell cytoplasm contains large secretory granules packed with tearproteins. Known tear proteins include: lysozyme, which plays a prominentbactericidal role on the corneal surface; lactoferrin, which functionsas both a bactericidal agent and as a potential inhibitor of complementactivation; secretory component, which regulates the transcellularmovement of IgA into acini lumen where it acts on the corneal surface toinhibit bacterial adhesion; and tear lipocalins (tear-specificprealbumin) and growth factors TGFα, TGFβ and EGF the functions of whichare not known. In rats, peroxidase is a tear component which has servedas a convenient marker in experimental studies. Tears not only have animportant bactericidal role, they also keep the cornea clean andlubricated and are important for the well-being of the cornealepithelium.

The surface of the eye is one of the most accessible and vulnerabletissues. Corneal epithelial cells confront environmental insultsconstantly including: UV irradiation, widely varying air temperaturefluxes, pollutants, bacteria and other microbial organisms. The tearfluid which bathes the corneal surface is the most likely source ofcytoprotective and anti-inflammatory agents since the cornea lacks bloodsupply, unlike other tissues where blood vessels supply such agents.Indeed, tear fluid is rich in bactericidal proteins. Dry Eye subjectssuffering insufficient tear production are subject to cornealulceration, infection or inflammation. Similar symptoms can be generatedby extended contact lens use, since volume of tear supply is limited.

When lacrimal acinar cell tear output is collectively deficient, ‘DryEye’ (also known as keratoconjunctivitis sicca [KCS]); is the result.Dry Eye is a common ocular manifestation of Sjogren's's syndrome, anautoimmune disease with unknown etiology that affects millions of peopleworldwide. Most commonly affected are post-menopausal women with varyingdegrees of severity. If untreated, Dry Eye can lead to corneal abrasion,ulceration, bacterial infection, and loss of vision. Molecularmechanisms underlying the pathogenic decline of secretory output by themain lacrimal gland are potentially multiple. Lacrimal glands ofSjogren's's syndrome subjects contain foci of B and T lymphocytes whosepathogenic expansion, possibly due to viral insult, can destroy lacrimalacini. However, acinar volume loss often appears insufficient relativeto the theoretical overcapacity of the main lacrimal gland. Estimatessuggest a potential secretory output up to ten-fold greater than isrequired to maintain a normal aqueous tear film layer. Other mechanismstherefore warrant attention, such as aberrant secretion of one orseveral common cytokines that may directly or indirectly alter lacrimalacinar cell function and/or lead to a decline in neural innervation.Novel autocrine/paracrine factor(s) released by lacrimal acinar cellsinto the tear film may be required for the health of the lacrimalsecretory machinery, ductal system, and corneal epithelium. Theperiacinar basement membrane is also required for normal secretoryfunction, in part via ‘BM180’ whose apparent synergy with laminin-1promotes stimulated tear secretion. Alteration of each of these factors,together or independent of hormonal changes, could contribute todecreased secretory capacity.

The lacrimal-corneal axis is a fundamental regulator of ocular healthand plays a key role in ocular surface inflammation associated with DryEye Syndromes and corneal injury. A host of mediators are implicated inthe development and progression of corneal inflammation, such as theproinflammatory cytokines TNF-α, IL-1β, IL-6 and the chemokine IL-8.Also involved are the arachidonic acid-derived eicosanoids which areproduced by the activity of cyclooxygenases (primarily PGE2),lipooxygenases (12 (s)-HETE) and cytochrome P450 (12 (r)-HETE).

Lacritin is a 12.3 kDa secreted glycoprotein that is apically releasedfrom human lacrimal acinar cells during reflex tearing and can bedetected in mixed reflex and basal human tears by ELISA and Westernblotting. Lacritin is also produced by corneal, conjunctival, meibomian,and salivary epithelia as one of the most eye-restricted genes. Recentstudies on lacritin mechanisms of action indicate converging PKCα andNFkB signaling pathways suggesting that lacritin may have a keyanti-inflammatory role on the ocular surface. Recent clinical studiessupport this hypothesis. Comparison of tear proteins from 19 subjectssuffering from Blepharitis (inflammation of the lid) vs 27 healthyvolunteers revealed lacritin to be decreased by 56% in subjects. Sumadreet al. (Invest Ophthalmol Vis Sci., 2011; 52:6265-6270;DOI:10.1167/iovs.10-6220) showed that lacritin acutely increased basaltearing to 30% over vehicle and that multiple doses per day were welltolerated. It was also recently reported that lacritin is selectivelydownregulated more than any other tear protein in contact lens-relateddry eye. Lacritin stimulates MUC16 production by human cornealepithelial cells at levels matching or exceeding that of serum (Laurie GE, et al. IOVS 2006; 47:ARVO E-Abstract 1606). Autologous serum is areportedly successful method of treating dry eye. Lacritin also promotesbasal tear secretion by cultured rat and monkey lacrimal acinar cellsand stimulates human corneal epithelial cell growth.

Few cell types appear capable of being targeted by lacritin. Targetedcells include lacrimal acinar, salivary ductal/HeLa, human corneal, andembryonic kidney cells, but no others among 17 different cell linestested. Its co-receptor syndecan-1 is widely expressed on ocular surfaceepithelia. Thus, lacritin appears to be a multifunctional eye-specificfactor with a potential role in tear secretion and corneal epithelialrenewal.

There is a long felt need in the art for compositions and methods usefulfor detecting and diagnosing dry eye, treating dry eye, and developingtreatment strategies and regimens based on the a diagnosis of dry eye.The present invention satisfies these needs.

SUMMARY

The present invention couples a novel mechanism for the molecularidentification of dry eye disease with a restorative therapy thataddresses cause. The invention relates to the discovery disclosed hereinthat a ˜90 KDa deglycanated form of syndecan-1 is abundant in tears ofnormal individuals but not in individuals suffering from dry eye.Furthermore a ˜25 kDa syndecan-1 fragment is detectable in dry, but notnormal tears. The invention also relates to the discovery that topicallacritin, the agonist of deglycanated syndecan-1, sensitizes cornealsensory nerves to drying of the surface of the eye, and increases theneural wet response. Accordingly, one embodiment of the presentinvention is directed to identifying dry eye by a relative decrease in˜90 kDa deglycanated form of syndecan-1 and/or the presence of 25 kDasyndecan-1 in tears. Another embodiment is directed to increasing thecorneal neural dry and wet responses by topical application of alacritin polypeptide to the eye.

Applicants have also discovered that that aqueous deficient dry eyetears are associated with decreased lacritin monomer, increasedlacritin-C splice variant, and latent (chronically active) heparanase(HPSE). Accordingly, in one embodiment a method is provided foridentifying patients suffering from dry eye and selecting such patientfor treatment. In one embodiment a method for identifying a subjecthaving dry eye is provided wherein the presence of at least one proteinselected from the group consisting of

-   -   latent heparanase;    -   90 kDa deglycanated SDC-1;    -   25 kDa SDC-1; and    -   inactive lacritin-C splice variant;        is detected in a tear sample obtained from the subject. Patients        with dry eye are then identified by those that have one or more        of the following:

a decreased level of latent heparanase and a corresponding increase inactive heparanase, relative to levels present in tears from a normaleye;

a decreased level of 90 kDa deglycanated SDC-1, relative to levelspresent in tears from a normal eye;

presence of 25 kDa SDC-1; and/or

presence of inactive lacritin-C splice variant. Such identified subjectscan then be treated by contacting the ocular surface of the subject'seyes with a composition comprising lacritin or a bioactive fragmentthereof.

The detection of latent heparanase, 90 kDa deglycanated SDC-1A, 25 kDaSDC-1 or inactive lacritin-C splice variant can be conducted usingstandard techniques known to those skilled in the art, including the useof antibodies. In one embodiment antibodies could be embedded inSchirmer strips on which tears are collected for precise and inexpensivemolecular diagnosis in an ophthalmologist's or optometrist's office.Current approaches for identifying subjects afflicted with dry eye donot address cause, and therefore suffer from inaccuracy and arenonspecific. Examples of current methods include: a) subjectquestionnaires, b) rose bengal or lissamine green staining of ocularsurface damage, c) Schirmer strip measurement of tear volume, d) tearbreak up time, e) tear evaporation rate, f) tear meniscus height orradius, g) tear film index or turnover rate, h) tear osmolarity, i)lysozyme or lactoferrin assay, and j) tear ferning analysis.

Restoration of active lacritin to the ocular surface has been found torescue the normal corneal sensory neural dry and wet responses necessaryfor normal eye physiology. Since all glands wetting the eye areregulated by the reflex are downstream of corneal sensory input,lacritin or lacritin fragments, synthetic peptides or mimetics shouldbenefit all forms of dry eye. Preclinical studies in rabbits and in dryeye mice models imply that it may also restore the density of cornealsensory innervation that decreases in dry eye. In contrast, commonlyused ‘artificial tears’ temporarily alleviate symptoms withoutaddressing cause.

It is disclosed herein that aqueous deficient dry eye tears areassociated with decreased lacritin monomer, increased lacritin-C splicevariant, less deglycanated SDC1, increased 25 kDa SDC1 fragment, anddecreased latent heparanase and increased active heparanase. Therefore,the present invention provides compositions and methods for detectingand diagnosing dry eye and for developing and providing treatmentregimens for subjects found to have dry eye using one or more of themarkers of dry eye disclosed herein. The present application providescompositions and methods for detecting and diagnosing dry eye, includingthe FOXO3 translocation assay disclosed herein. Multiple methods arealso available and described for detecting and measuring the protein andprotein fragments useful for detecting and diagnosing eye.

The present invention further provides for the use of lacritin, orbiologically active fragments or homologs thereof, to sensitize cornealsensory nerves to drying of the surface of the eye and increases theneural wet response. In one embodiment, use of topical lacritin orfragment N-94 (SEQ ID NO: 7) restores or increases tearing. In oneaspect, the use restores basal tearing. In one aspect, topicaladministration of lacritin suppresses lacrimal gland inflammation.

In accordance with one embodiment a method of treatment is provided torestore the levels of 90 kDa syndecan-1 (SDC1) or other deglycanatedforms of syndecan-1 in the tears of a subject with dry eye. RestoringSDC1 enhances activity of lacritin that is present. The presentinvention further provides methods of treating dry eye using inhibitorsof transglutaminase (TGM), which can be inhibitors of TGM activity orlevels or synthesis.

In accordance with one embodiment, a composition is provided comprisinga peptide, a non-native peptide, or a peptidomimetic derivative,comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or asequence that differs from SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7 and SEQ ID NO: 8 by 1, 2, 3, 4 or 5 amino acids, or abiologically active fragment, homolog, or derivative thereof. In oneembodiment a peptide differs from SEQ ID NO: 1, SEQ ID NO: 5, or SEQ IDNO: 7 by 1, 2, 3, 4 or 5 conservative amino acid substitutions. In oneembodiment, the amino acid modifications are amino acid substitution,and in one embodiment the substitutions are conservative amino acidsubstitutions.

In some embodiments, the peptide of the present disclosure comprises anamino acid sequence which has at least 75%, 80%, 85%, 90% or 95%sequence identity to amino acid sequence SEQ ID NO: 1, SEQ ID NO: 5, orSEQ ID NO: 7 or a biologically active fragment, homolog, or derivativethereof.

In some embodiments, the peptide of the present disclosures comprises anon-native amino acid sequence which has at least 75%, 80%, 85%, 90% or95% sequence identity to amino acid sequence SEQ ID NO: 1, SEQ ID NO: 5,or SEQ ID NO: 7 or a peptidomimetic derivative of SEQ ID NO: 1, SEQ IDNO: 5 or SEQ ID NO: 7. The statement that the peptide is a non-native isintended to exclude the native peptides of parent lacritin proteins.

In accordance with one embodiment a method of enhancing corneal woundhealing in a subject in need thereof is provided. The method comprisescontacting an ocular surface of said subject with a compositioncomprising lacritin or a bioactive fragment thereof. In one embodimentthe bioactive fragment of lacritin is selected from the group consistingof

KQFIENGSEFAQKLLKKFS (SEQ ID NO: 5);

KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7);

KQFIENGSEFANKLLKKFS (SEQ ID NO: 6); and

KQFIENGSEFANKLLKKFSLLKPWA (SEQ ID NO: 8) or a derivative thereof thatdiffers from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 byone or two amino acid substitutions. In one embodiment the subject isrecovering from PRK (photorefractive keratectomy) or LASIK(Laser-Assisted in situ Keratomileusis) surgery.

In another embodiment a bactericidal composition is provided, comprisinga C-terminal fragment of lacritin. In one embodiment the fragment is apeptide selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQID NO: 8 or a derivative thereof that differs from SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 by one or two amino acidsubstitutions. In one embodiment the fragment is a peptide consisting ofthe sequence of SEQ ID NO: 7. In one embodiment the compositioncomprises a pharmaceutically acceptable carrier wherein the compositionis suitable for topical administration to an ocular surface of asubject.

In one embodiment the composition, further comprises a secondanti-bacterial agent. As disclosed herein a method of treating a cornealinfection is provided wherein the method comprises contacting the corneaof a subject in need thereof with the composition comprising aC-terminal fragment of lacritin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Detection of rare, deglycanated and 25 kDa forms of syndecan-1respectively in normal and dry eye tears. Left paired samples (beforephotorefractive keratectomy (PRK) or Laser-Assisted in situKeratomileusis (LASIK)) ˜90 kDa deglycanated SDC1 is abundant in normaltears and barely detectable in dry eye tears. Day 1 (D1) after PRK orLASIK surgery, ˜90 kDa deglycanated SDC1 is less than in dry eye tears.Also, a 25 kDa SDC1 fragment is apparent in dry eye tears. Surgerypromotes dry eye by severing corneal sensory nerves. Week 1 (W1) afterPRK or LASIK surgery, the ˜90 deglycanated SDC1 level is restored innormal tears that received surgery, and 25 kDa level in dry eye tearsremains elevated (less at 1 Month (M1).

FIGS. 2A & 2B. Detection of elevated levels of inactive lacritin-Csplice variant in dry eye tears. FIG. 2A is a Western blot demonstratingthe detection of lacritin-C using mab 4F6. Lacritin-C splice variant isconstantly elevated in dry eye. FIG. 2B is a Western blot usingsecondary antibody alone and serves as a negative control. No bands aredetected using only the secondary antibody.

FIGS. 3A & 3B. Detection of more latent heparanase in normal tears vsdry eye tears, and more activated heparanase in dry eye years. FIG. 3Ais a Western blot demonstrating the detection of heparanase using #1453antibody. Latent heparanase is indicated by ˜75 kDa band; activeheparanase indicated by ˜50 kDa band. FIG. 3B is a Western blotdemonstrating the detection of heparanase using #753 antibody.

FIGS. 4A-4C: Corneal health restorative activity of lacritin, and aC-terminal 25 amino acid fragment of lacritin (LACRIPEP). Cultured humancorneal epithelial cells were treated with inflammatory cytokines toinduce stress, and cells were treated with 10 nM of an inactive lacritintruncation mutant (C-25), lacritin or LACRIPEP. Measurements ofcytoplasmic staining in a FOXO3 assay (wherein nuclear FOXO3 staining isindicative of cell death) reveal LACRIPEP is equally active as lacritin(See FIG. 4A) in enhancing cell survival relative to the negativecontrol (C-25). Studies in dry eye (Aire−/−) mice also demonstrate thebioactivity of topically administered LACRIPEP. LACRIPEP prevents lossof tearing as dry eye disease develops in Aire(−/−) dry eye mice (FIG.4B; closed circles) relative to topically administered PBS (openedcircles) and Aire(−/−) dry eye mice administered LACRIPEP have lesscorneal staining, which is an indicator of cell death, as dry eyedisease develops (FIG. 4C; closed circles) relative to PBS (openedcircles).

FIG. 5 Comparative pro-survival activity of lacritin and lacritinsynthetic peptides. Quantitation of FOXO3 immunostaining in interferon-γand tumor necrosis factor stressed human HCE-T cells treated withlacritin C-terminal truncation mutant C-25 (negative control; inactive),lacritin (lacrt), lacritin C-terminal peptide N-94 (SEQ ID NO: 7) orN-94/C-6 (SEQ ID NO: 5), or tissue transglutaminase polymerized lacritin(inactive). Dosage for each administered peptide is 10 nM. More nuclearstaining indicates stress/death. More cytoplasmic staining (arrows)indicates survival. 203-379 cells were counted for each treatment.Comparison of all but polymerized lacrt vs C-25 by two-way ANOVA withBonferroni post test, P=0.01.

FIGS. 6A & 6B LACRIPEP shows surprising stability in human tears. FIG.6A presents immunoblots of protease sensitive positive control ‘SN pep’from a different protein, and LACRIPEP (‘N-94’; SEQ ID NO: 7), afterincubation in lacritin-depleted human tears for 2-16 hr at 37° C. FIG.6B presents mass spectrometric analysis, wherein the top row presents MSprofiles of SN pep, LACRIPEP (‘N-94’), and LACRIPEP without sixC-terminal amino acids (‘N-94/C-6’) prior to addition to tears and the37° C. incubation step, and the bottom row provides MS profiles afterincubation in lacritin depleted tears for 4 hr at 37° C.

FIGS. 7A & 7B Biphasic dose response of LACRIPEP. Biphasic dose responseof topical LACRIPEP was demonstrated testing rabbit basal tearing (FIG.7A) and in rat corneal sensory nerve stimulation (pLAC; FIG. 7B)relative to an inactive lacritin fragment control (C-25D).

FIG. 8 Distribution of a single 4 μM dose of topical ¹²⁵I-Lacripep-Y onrat eyes. Slight amounts of ¹²⁵I-Lacripep-Y are detectable in blood andplasma. A considerable amount is retained in tears.

FIG. 9 Alignment of the 25 amino acid C-terminal fragments of lacritinhomologs from primate species including human (SEQ ID NO: 7); Chimpanzee(SEQ ID NO: 17); Bushbaby (SEQ ID NO: 18); Gorilla (SEQ ID NO: 19);Macaque (SEQ ID NO: 20); Marmoset (SEQ ID NO: 21); Mouse Lemur (SEQ IDNO: 22) and Orangutan (SEQ ID NO: 23) demonstrating a high sequenceconservation between primate species.

DETAILED DESCRIPTION Abbreviations and Acronyms

FACS means fluorescence activated cell sorter

HCE means human corneal epithelial

HPSE means heparanase

HS means heparan sulfate

HSG means human salivary gland

INFG means interferon gamma (also referred to as IFNG)

IRB means institutional review board

SDC1 means syndecan-1

TGM means transglutaminase

TNF means tumor necrosis factor

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “lacritin polypeptide” and the like terms isdefined as any peptide comprising the amino acid sequence SEQ ID NO: 1and or a biologically active fragment, homolog, or derivative thereof.As used herein, the term “biologically active fragments” or “bioactivefragment” of a lacritin polypeptide encompasses natural or syntheticportions of the amino acid sequenceMKFTTLLFLAAVAGALVYAEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 1). Fragments of lacritin(SEQ ID NO: 1) include, for example: KQFIENGSEFAQKLLKKFS (SEQ ID NO: 5)(‘N-94/C-6’) (Wang et al., (2006) J. Cell Biol. 174, 689-700). andKQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7) (‘N-94’) (see Zhang et al.,(2013) J. Biol. Chem. 288, 12090-12101).

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical value or range, it modifies that range by extending theboundaries above and below the numerical values set forth. For example,in one aspect, the term “about” is used herein to modify a numericalvalue above and below the stated value by a variance of 20%, but is notintended to designate any value or range of values to only this broaderdefinition. Each value or range of values preceded by the term “about”is also intended to encompass the embodiment of the stated absolutevalue or range of values.

As used herein an “acylated” amino acid is an amino acid comprising anacyl group which is non-native to a naturally-occurring amino acid,regardless by the means by which it is produced. Exemplary methods ofproducing acylated amino acids and acylated peptides are known in theart and include acylating an amino acid before inclusion in the peptideor peptide synthesis followed by chemical acylation of the peptide. Insome embodiments, the acyl group causes the peptide to have one or moreof (i) a prolonged half-life in circulation, (ii) a delayed onset ofaction, (iii) an extended duration of action, and (iv) an improvedresistance to proteases.

As used herein, an “alkylated” amino acid is an amino acid comprising analkyl group which is non-native to a naturally-occurring amino acid,regardless of the means by which it is produced. Exemplary methods ofproducing alkylated amino acids and alkylated peptides are known in theart and including alkylating an amino acid before inclusion in thepeptide or peptide synthesis followed by chemical alkylation of thepeptide. Without being held to any particular theory, it is believedthat alkylation of peptides will achieve similar, if not the same,effects as acylation of the peptides, e.g., a prolonged half-life incirculation, a delayed onset of action, an extended duration of action,and an improved resistance to proteases.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

As used herein, the term “hydrophilic moiety” refers to any compoundthat is readily water-soluble or readily absorbs water, and which aretolerated in vivo by mammalian species without toxic effects (i.e. arebiocompatible). Examples of hydrophilic moieties include polyethyleneglycol (PEG), polylactic acid, polyglycolic acid, apolylactic-polyglycolic acid copolymer, polyvinyl alcohol,polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline,polyhydroxyethyl methacrylate, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatised cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose and co-polymersthereof, as well as natural polymers including, for example, albumin,heparin and dextran.

A “subject” of experimentation, diagnosis or treatment is an animal,including a human.

As used herein the term “Dry eye” (or Dry Eye) encompasses any conditionin which there are insufficient tears to lubricate and nourish the eye.Subjects with dry eyes either do not produce enough tears or have a poorquality of tears. Dry eye as used herein includes, but is not limitedto: aqueous-deficient and evaporative dry eye. Aqueous-deficient dry eyeincludes, but is not limited to, Sjogren's Syndrome Dry Eye (includingprimary and secondary), Non-Sjogren's Dry Eye (including lacrimaldeficiency, lacrimal gland duct obstruction, reflex block, and fromsystemic drugs). Evaporative dry eye includes, but is not limited to,Intrinsic (including meibomian oil deficiency, disorders of the lidaperture, low blink rate, and resulting from the drug action ofAccutane) and Extrinsic (including Vitamin A deficiency, topical drugpreservatives, contact lens wear, and ocular surface disease (such asallergies).

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating dry eye” willrefer in general to maintaining basal tear levels near normal levels andmay include increasing tear levels depending on a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a pharmaceutical agent refers to a nontoxic but sufficientamount of an agent to provide the desired effect. For example onedesired effect would be the prevention or treatment of dry eye. Theamount that is “effective” will vary from subject to subject, dependingon the age and general condition of the individual, mode ofadministration, and the like. Thus, it is not always possible to specifyan exact “effective amount.” However, an appropriate “effective” amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation.

The terms “additional therapeutically active compound” or “additionaltherapeutic agent”, as used in the context of the present invention,refers to the use or administration of a compound for an additionaltherapeutic use for a particular injury, disease, or disorder beingtreated. Such a compound, for example, could include one being used totreat an unrelated disease or disorder, or a disease or disorder whichmay not be responsive to the primary treatment for the injury, diseaseor disorder being treated.

The term “identity” as used herein relates to the similarity between twoor more sequences. Identity is measured by dividing the number ofidentical residues by the total number of residues and multiplying theproduct by 100 to achieve a percentage. Thus, two copies of exactly thesame sequence have 100% identity, whereas two sequences that have aminoacid/nucleic acid deletions, additions, or substitutions relative to oneanother have a lower degree of identity. Those skilled in the art willrecognize that several computer programs, such as those that employalgorithms such as BLAST (Basic Local Alignment Search Tool, Altschul etal. (1993) J. Mol. Biol. 215:403-410) are available for determiningsequence identity.

As used herein an amino acid “modification” refers to a substitution ofan amino acid, or the derivation of an amino acid by the addition and/orremoval of chemical groups to/from the amino acid, and includessubstitution with any of the 20 amino acids commonly found in humanproteins, as well as atypical or non-naturally occurring amino acids.Commercial sources of atypical amino acids include Sigma-Aldrich(Milwaukee, Wis.), ChemPep Inc. (Miami, Fla.), and GenzymePharmaceuticals (Cambridge, Mass.). Atypical amino acids may bepurchased from commercial suppliers, synthesized de novo, or chemicallymodified or derivatized from naturally occurring amino acids.

As used herein an amino acid “substitution” refers to the replacement ofone amino acid residue by a different amino acid residue.

As used herein, the term “conservative amino acid substitution” isdefined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

-   -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;

III. Polar, positively charged residues:

-   -   His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

-   -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp, acetyl phenylalanine

The term “isolated” as used herein means having been removed from itsnatural environment. In some embodiments, a peptide is made throughrecombinant methods and the peptide is isolated from the host cell.

The term “purified,” as defined herein means the isolation of a moleculeor compound in a form that is substantially free of contaminantsnormally associated with the molecule or compound in a native or naturalenvironment and means having been increased in purity as a result ofbeing separated from other components of the original composition. Theterm “purified polypeptide” is used herein to describe a polypeptidewhich has been separated from other compounds including, but not limitedto nucleic acid molecules, lipids and carbohydrates.

A “peptidomimetic” refers to a chemical compound having a structure thatis different from the general structure of an existing peptide, but thatfunctions in a manner similar to the existing peptide, e.g., bymimicking the biological activity of that peptide. Peptidomimeticstypically comprise naturally-occurring amino acids and/or unnaturalamino acids, but can also comprise modifications to the peptidebackbone. For example a peptidomimetic may include a sequence ofnaturally-occurring amino acids with the insertion or substitution of anon-peptide moiety, e.g. a retroinverso fragment, or incorporation ofnon-peptide bonds such as an azapeptide bond (CO substituted by NH) orpseudo-peptide bond (e.g. NH substituted with CH₂), or an ester bond(e.g., depsipeptides, wherein one or more of the amide (—CONHR—) bondsare replaced by ester (COOR) bonds). Alternatively the peptidomimeticmay be devoid of any naturally-occurring amino acids.

As use herein, the terms “administration of” and or “administering” acompound should be understood to mean providing a compound of theinvention to a subject in need of treatment.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

As used herein the term “amino acid” encompasses any molecule containingboth amino and carboxyl functional groups, wherein the amino andcarboxylate groups are attached to the same carbon (the alpha carbon).The alpha carbon optionally may have one or two further organicsubstituents. For the purposes of the present disclosure designation ofan amino acid without specifying its stereochemistry is intended toencompass either the L or D form of the amino acid, or a racemicmixture. However, in the instance where an amino acid is designated byits three letter code and includes a superscript number, the D form ofthe amino acid is specified by inclusion of a lower case d before thethree letter code and superscript number (e.g., dLys1), wherein thedesignation lacking the lower case d (e.g., Lys1) is intended to specifythe native L form of the amino acid. In this nomenclature, the inclusionof the superscript number designates the position of the amino acid inthe peptide sequence numbered consecutively from the N-terminus. Theexpression “amino acid” as used herein is meant to include both naturaland synthetic amino acids, and both D and L amino acids. “Standard aminoacid” means any of the twenty L-amino acids commonly found in naturallyoccurring peptides.

As used herein the term “non-coded amino acid” encompasses any aminoacid that is not an L-isomer of any of the following 20 amino acids:Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, Tyr, regardless of whether it is preparedsynthetically or derived from a natural source.

As used herein a general reference to a peptide is intended to encompasspeptides that have modified amino and carboxy termini, including but notlimited to salts. For example, an amino acid sequence designating thestandard amino acids is intended to encompass standard amino acids atthe N- and C-terminus as well as a corresponding hydroxyl acid at theN-terminus and/or a corresponding C-terminal amino acid modified tocomprise an amide group in place of the terminal carboxylic acid Aminoacids contained within the peptides of the present invention, andparticularly at the carboxy- or amino-terminus, can be modified bymethylation, amidation, acetylation or substitution with other chemicalgroups which can change the peptide's circulating half-life withoutadversely affecting their activity. Additionally, a disulfide linkagemay be present or absent in the peptides of the invention.

The term “amino acid” is used interchangeably with “amino acid residue,”and may refer to a free amino acid and to an amino acid residue of apeptide. It will be apparent from the context in which the term is usedwhether it refers to a free amino acid or a residue of a peptide.

Amino acids may be classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

The nomenclature used to describe the peptide compounds of the presentinvention follows the conventional practice wherein the amino group ispresented to the left and the carboxy group to the right of each aminoacid residue. In the formulae representing selected specific embodimentsof the present invention, the amino- and carboxy-terminal groups,although not specifically shown, will be understood to be in the formthey would assume at physiologic pH values, unless otherwise specified.

The term “basic” or “positively charged” amino acid as used herein,refers to amino acids in which the R groups have a net positive chargeat pH 7.0, and include, but are not limited to, the standard amino acidslysine, arginine, and histidine.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as singlechain antibodies and humanized antibodies.

An “antimicrobial agent,” as used herein, refers to any compound whichimpedes the growth of any microbes, or kills such microbes.

A “bactericidal agent,” as used herein, refers to any compound whichimpedes the growth of bacteria, or kills bacteria.

As used herein, the term “biologically active fragments” or “bioactivefragment” of the polypeptides encompasses natural or synthetic portionsof the full length protein that are capable of specific binding to theirnatural ligand or of performing the function of the protein.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base pairing rules. For example, for the sequence “AGT,”is complementary to the sequence “TCA.”

The use of the word “detect” and its grammatical variants refers tomeasurement of the species without quantification, whereas use of theword “determine” or “measure” with their grammatical variants are meantto refer to measurement of the species with quantification. The terms“detect” and “identify” are used interchangeably herein.

As used herein, a “detectable marker” or a “reporter molecule” is anatom or a molecule that permits the specific detection of a compoundcomprising the marker in the presence of similar compounds without amarker. Detectable markers or reporter molecules include, e.g.,radioactive isotopes, antigenic determinants, enzymes, nucleic acidsavailable for hybridization, chromophores, fluorophores,chemiluminescent molecules, electrochemically detectable molecules, andmolecules that provide for altered fluorescence polarization or alteredlight scattering.

As used herein, the phrase “enhancing survival” refers to decreasing theamount of death, or the rate of death, in a cell population. Enhancingsurvival can be due to preventing cell death alone (e.g., cell death inconjunction with apoptosis), or decreasing the rate of cell death. Thedecrease in cell death can also result from indirect effects such asinducing proliferation of some cells, such indirect effect effectivelyreplenishing at least some or all of a population of cells as they dieEnhancing survival of cells can also be accomplished by a combination ofinducing proliferation and decreasing cell death, or the rate of celldeath. “Promoting survival” and “enhancing survivability” are usedinterchangeably with “enhancing survival” herein.

A “fragment” or “segment” is a portion of an amino acid sequence,comprising at least one amino acid, or a portion of a nucleic acidsequence comprising at least one nucleotide. The terms “fragment” and“segment” are used interchangeably herein. A fragment of a lacritinpeptide which is used herein as part of a composition for use in atreatment or to elicit a lacritin effect is presumed to be abiologically active fragment for the response to be elicited.

As used herein, a “functional” biological molecule is a biologicalmolecule in a form in which it exhibits a property or activity by whichit is characterized. A functional enzyme, for example, is one whichexhibits the characteristic catalytic activity by which the enzyme ischaracterized.

As used herein, a “gene” refers to the nucleic acid coding sequence aswell as the regulatory elements necessary for the DNA sequence to betranscribed into messenger RNA (mRNA) and then translated into asequence of amino acids characteristic of a specific polypeptide.

As used herein, the term “insult” refers to contact with a substance orenvironmental change that results in an alteration of normal cellularmetabolism in a cell or population of cells. Environmental insults mayinclude, but are not limited to, chemicals, environmental pollutants,heavy metals, viral or bacterial infections, changes in temperature,changes in pH, as well as agents producing oxidative damage, DNA damage,or pathogenesis. The term “insult” is used interchangeably with“environmental insult” herein.

As used herein, the term “syndecan-1” refers to peptides comprising theamino acid sequence of SEQ ID NO: 2 and biologically active fragments,derivatives, and homologs thereof. As used herein, the term“biologically active fragments” or “bioactive fragment” of a syndecan-1polypeptide encompasses natural or synthetic portions of the amino acidsequence

(SEQ ID NO: 2) MRRAALWLWLCALALSLQPALPQIVATNLPPEDODGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKEVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQ KPTKQEEFYA.The underlined portion of SEQ ID NO: 2 represents “shed and deglycanated90 kDa form of syndecan-1” (or 90 kDa deglycanated SDC-1), having thesequence:

(SEQ ID NO: 3) QIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKE.

As used herein, the term “a 25 kDa fragment of SDC-1 ectodomain” (or 25kDa SDC-1) refers to a 25 kDa fragment of SDC-1 that contains the LPEVsequence of the 90 kDa deglycanated SDC-1.

As used herein, the term “heparanase” refers to peptides comprising theamino acid sequence of SEQ ID NO: 4 and biologically active fragments,derivatives, and homologs thereof. As used herein, the term“biologically active fragments” or “bioactive fragment” of a heparanasepolypeptide encompasses natural or synthetic portions of the amino acidsequence

(SEQ ID NO: 4) MLLRSKPALPPPLMLLLLGPLGPLSPGALPRPAQAQDVVDLDFFTQEPLHLVSPSFLSVTIDANLATDPRFLILLGSPKLRTLARGLSPAYLRFGGTKTDFLIFDPKKESTFEERSYWQSQVNQDICKYGSIPPDVEEKLRLEWPYQEQLLLREHYQKKFKNSTYSRSSVDVLYTFANCSGLDLIFGLNALLRTADLQWNSSNAQLLLDYCSSKGYNISWELGNEPNSFLKKADIFINGSQLGEDFIQLHKLLRKSTFKNAKLYGPDVGQPRRKTAKMLKSFLKAGGEVIDSVTWHHYYLNGRTATKEDFLNPDVLDIFISSVQKVFQVVESTRPGKKVWLGETSSAYGGGAPLLSDTFAAGFMWLDKLGLSARMGIEVVMRQVFFGAGNYHLVDENFDPLPDYWLSLLFKKLVGTKVLMASVQGSKRRKLRVYLHCTNTDNPRYKEGDLTLYAINLHNVTKYLRLPYPFSNKQVDKYLLRPLGPHGLLSKSVQLNGLTLKMVDDQTLPPLMEKPLRPGSSLGLPAFSYSFFVIRNAKVAACI.

As used herein, a “ligand” is a compound that specifically binds to atarget compound. A ligand (e.g., an antibody) “specifically binds to” or“is specifically immunoreactive with” a compound when the ligandfunctions in a binding reaction which is determinative of the presenceof the compound in a sample of heterogeneous compounds. Thus, underdesignated assay (e.g., immunoassay) conditions, the ligand bindspreferentially to a particular compound and does not bind to asignificant extent to other compounds present in the sample. Forexample, an antibody specifically binds under immunoassay conditions toan antigen bearing an epitope against which the antibody was raised. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular antigen. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with an antigen. See Harlow andLane, 1988, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

As used herein, the term “linkage” refers to a connection between twogroups. The connection can be either covalent or non-covalent, includingbut not limited to ionic bonds, hydrogen bonding, andhydrophobic/hydrophilic interactions.

As used herein, the term “linker” refers to a molecule that joins twoother molecules either covalently or noncovalently, e.g., through ionicor hydrogen bonds or van der Waals interactions.

“Ocular surface,” as used herein, refers to the surface of the eye,particularly the corneal surface.

The phrase “ocular surface-associated disease, disorder, or condition,”as used herein, refers to any disease, disorder or condition whichdirectly or indirectly causes, or can cause, any of the problems orsymptoms described herein regarding disease, disorders, or conditions ofthe ocular surface.

“Operably linked” refers to a juxtaposition wherein the components areconfigured so as to perform their usual function. Thus, controlsequences or promoters operably linked to a coding sequence are capableof effecting the expression of the coding sequence.

A “marker” is an atom or molecule that permits the specific detection ofa molecule comprising that marker in the presence of similar moleculeswithout such a marker. Markers include, for example radioactiveisotopes, antigenic determinants, nucleic acids available forhybridization, chromophors, fluorophors, chemiluminescent molecules,electrochemically detectable molecules, molecules that provide foraltered fluorescence polarization or altered light scattering andmolecules that allow for enhanced survival of an cell or organism (i.e.a selectable marker). A reporter gene is a gene that encodes for amarker.

The term “measuring the level of expression” or “determining the levelof expression” as used herein refers to any measure or assay which canbe used to correlate the results of the assay with the level ofexpression of a gene or protein of interest. Such assays includemeasuring the level of mRNA, protein levels, etc. and can be performedby assays such as northern and western blot analyses, binding assays,immunoblots, etc. The level of expression can include rates ofexpression and can be measured in terms of the actual amount of an mRNAor protein present. Such assays are coupled with processes or systems tostore and process information and to help quantify levels, signals, etc.and to digitize the information for use in comparing levels.

A “polylinker” is a nucleic acid sequence that comprises a series ofthree or more different restriction endonuclease recognitions sequencesclosely spaced to one another (i.e. less than 10 nucleotides betweeneach site).

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner

A “constitutive promoter is a promoter which drives expression of a geneto which it is operably linked, in a constant manner in a cell. By wayof example, promoters which drive expression of cellular housekeepinggenes are considered to be constitutive promoters.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living cell substantiallyonly when an inducer which corresponds to the promoter is present in thecell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

As used herein, “nucleic acid,” “DNA,” and similar terms also includenucleic acid analogs, i.e. analogs having other than a phosphodiesterbackbone. For example, the so called “peptide nucleic acids,” which areknown in the art and have peptide bonds instead of phosphodiester bondsin the backbone, are considered within the scope of the presentinvention.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “peptide” encompasses a sequence of 3 or more amino acidswherein the amino acids are naturally occurring or synthetic(non-naturally occurring) amino acids. Peptide mimetics include peptideshaving one or more of the following modifications:

1. peptides wherein one or more of the peptidyl —C(O)NR— linkages(bonds) have been replaced by a non-peptidyl linkage such as a—CH2-carbamate linkage (—CH2OC(O)NR—), a phosphonate linkage, a—CH2-sulfonamide (—CH 2-S(O)2NR—) linkage, a urea (—NHC(O)NH—) linkage,a —CH2-secondary amine linkage, or with an alkylated peptidyl linkage(—C(O)NR—) wherein R is C1-C4 alkyl;

2. peptides wherein the N-terminus is derivatized to a —NRR1 group, to a—NRC(O)R group, to a —NRC(O)OR group, to a —NRS(O)2R group, to a—NHC(O)NHR group where R and R1 are hydrogen or C1-C4 alkyl with theproviso that R and R1 are not both hydrogen;

3. peptides wherein the C terminus is derivatized to —C(O)R2 where R 2is selected from the group consisting of C1-C4 alkoxy, and —NR3R4 whereR3 and R4 are independently selected from the group consisting ofhydrogen and C1-C4 alkyl.

Synthetic or non-naturally occurring amino acids refer to amino acidswhich do not naturally occur in vivo but which, nevertheless, can beincorporated into the peptide structures described herein. The resulting“synthetic peptide” contain amino acids other than the 20 naturallyoccurring, genetically encoded amino acids at one, two, or morepositions of the peptides. For instance, naphthylalanine can besubstituted for tryptophan to facilitate synthesis. Other syntheticamino acids that can be substituted into peptides includeL-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alpha-amino acids such asL-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha.-methylalanyl,beta-amino acids, and isoquinolyl. D amino acids and non-naturallyoccurring synthetic amino acids can also be incorporated into thepeptides. Other derivatives include replacement of the naturallyoccurring side chains of the 20 genetically encoded amino acids (or anyL or D amino acid) with other side chains.

The term “fusion polypeptide” or “fusion protein” refers to a chimericprotein containing a reference protein (e.g., lacritin) joined at the N-and/or C-terminus to one or more heterologous sequences (e.g., a nonlacritin polypeptide, such as syndecan). Polypeptide molecules are saidto have an “amino terminus” (N terminus) and a “carboxy terminus” (Cterminus) because peptide linkages occur between the backbone aminogroup of a first amino acid residue and the backbone carboxyl group of asecond amino acid residue. The terms “N terminal” and “C terminal” inreference to polypeptide sequences refer to regions of polypeptidesincluding portions of the N terminal and C terminal regions of thepolypeptide, respectively. A sequence that includes a portion of the Nterminal region of polypeptide includes amino acids predominantly fromthe N terminal half of the polypeptide chain, but is not limited to suchsequences. For example, an N terminal sequence may include an interiorportion of the polypeptide sequence including bases from both the Nterminal and C terminal halves of the polypeptide. The same applies to Cterminal regions. N terminal and C terminal regions may, but need not,include the amino acid defining the ultimate N terminus and C terminusof the polypeptide, respectively.

The fusion proteins of the invention may be prepared by recombinantmethods or by solid phase chemical peptide synthesis methods. Suchmethods have been known in the art since the early 1960's (Merrifield,1963) (See also Stewart et al., Solid Phase Peptide Synthesis, 2 ed.,Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recently beenemployed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). In addition, a numberof available FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 431A automated peptidesynthesizer. Such equipment provides ready access to the peptides of theinvention, either by direct synthesis or by synthesis of a series offragments that can be coupled using other known techniques.

The invention also includes a stable cell line that expresses a lacritinbioactive fragment or a lacritin/syndecan-1 fusion protein, as well asan expression cassette comprising a nucleic acid molecule encoding thelacritin fragment or lacritin/syndecan-1 fusion protein, and a vectorcapable of expressing the nucleic acid molecule of the invention in ahost cell. Preferably, the expression cassette comprises a promoter,e.g., a constitutive or regulatable promoter, operably linked to thenucleic acid sequence. In one embodiment, the expression cassettecontains an inducible promoter. Also provided is a host cell, e.g., aprokaryotic cell or an eukaryotic cell such as a plant or vertebratecell, e.g., a mammalian cell, including but not limited to a human,non-human primate, canine, feline, bovine, equine, ovine or rodent(e.g., rabbit, rat, ferret or mouse) cell, which comprises theexpression cassette or vector of the invention, and a kit whichcomprises the nucleic acid molecule, expression cassette, vector, hostcell or lacritin/syndecan-1 fusion protein.

A “vector” is also meant to include a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “vector” includes an autonomouslyreplicating plasmid or a virus. The term should also be construed toinclude non-plasmid and non-viral compounds which facilitate transfer ofnucleic acid into cells, such as, for example, polylysine compounds,liposomes, and the like. Examples of viral vectors include, but are notlimited to, adenoviral vectors, adeno-associated virus vectors,retroviral vectors, plasmids, cosmids, lambda phage vectors, and thelike.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

As used herein, the term “wound” relates to a physical tear or ruptureto a tissue or cell layer. A wound may occur by any physical insult,including a surgical procedure.

EMBODIMENTS

As disclosed herein compositions having lacritin based activity aredisclosed for treating an ocular surface-associated disease, disorder,or condition. In accordance with one embodiment a composition isprovided comprising a lacritin polypeptide, a bioactive fragment oflacritin, a non-native lacritin peptide, or peptidomimetic derivative oflacritin. In one embodiment the composition comprises a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 5, and SEQ ID NO: 6 or a sequence that differs fromSEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 5, or SEQ ID NO: 6by 1, 2, 3, 4 or 5 amino acid modifications. In one embodiment thecomposition comprises a sequence selected from the group consisting ofSEQ ID NO: 7 or SEQ ID NO: 8 or a sequence that differs from SEQ ID NO:7 or SEQ ID NO: 8 by 1, 2, 3, 4 or 5 amino acid substitutions, and in afurther embodiment the 1, 2, 3, 4 or 5 amino acid substitutions areconservative amino acid substitutions.

In one embodiment a composition is provided comprising a bioactivefragment of lacritin, wherein the bioactive fragment consists of thesequence of SEQ ID NO: 7 or a derivative that differs from SEQ ID NO: 7by a single amino acid substitution. In one embodiment the single aminoacid substitution is a conservative amino acid substation and in afurther embodiment the amino acid substitution is at position 4, 6, 8,10, 17 and 19. In one embodiment the bioactive fragment consists of thesequence of SEQ ID NO: 7 or a derivative that differs from SEQ ID NO: 7by a single amino acid substitution at position 4 or 19. Surprisingly,applicants have found that the 25 amino acid C-terminal fragment ofnative human lacritin (SEQ ID NO: 7) has enhance stability in humantears relative to the same fragment having the terminal 6 amino acidsremoved (SEQ ID NO: 5). In particular, immunoblotting reveals thatN-94/C-6 loses epitopes after incubation in lacritin depleted tears for4 hr at 37° C. whereas Lacripep (‘N-94’; SEQ ID NO: 7) does not.

Although topical application of ophthalmic products has remained themost popular and well-tolerated administration route for patientcompliance, the bioavailability of eye drops is severely hindered byblinking, baseline and reflex lacrimation, and nasolacrimal drainage.One solution to enhance the therapeutic index of topical treatments isthrough the application of polymeric nanoparticles as drug carriers. Inaccordance with one embodiment a pharmaceutical composition is providedcomprising lacritin, or a bioactive fragment thereof linked to ananoparticle. In one embodiment the nanoparticle is a thermo-responsiveelastin-like polypeptide (ELP). ELPs are composed of the repetitivepentapeptide motif (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 24) and exhibitunique reversible inverse phase transition temperatures, Tt, below whichthey solubilize and above which they phase separate. In one embodimentthe carboxy terminus of lacritin or a bioactive fragment thereof islinked to an ELP and in one embodiment the C-terminus of a peptideconsisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8is linked to the repetitive pentapeptide motif (VPGSG)₄₈(VPGIG)₄₈ (SEQID NO: 28).

In accordance with one embodiment, a composition is provided comprisinga syndecan-1 peptide, a non-native peptide, or a peptidomimeticderivative thereof. In one embodiment the peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3 or asequence that differs from SEQ ID NO: 2 and SEQ ID NO: 3 by 1, 2, 3, 4or 5 amino acids, and homologs and fragments thereof. In one embodimenta peptide differs from SEQ ID NO: 2 and SEQ ID NO: 3 by 1, 2, 3, 4 or 5conservative amino acid substitutions. In one embodiment, the amino acidmodifications are amino acid substitution, and in one embodiment thesubstitutions are conservative amino acid substitutions. In oneembodiment the composition comprises a syndecan-1 fragment consisting ofthe sequence of SEQ ID NO: 2.

In some embodiments, the peptide of the present disclosure comprises anamino acid sequence which has at least 75%, 80%, 85%, 90% or 95%sequence identity to an amino acid sequence of SEQ ID NO: 2 or SEQ IDNO: 3 or a fragment or homolog thereof.

In some embodiments, the peptide of the present disclosures comprises anon-native amino acid sequence which has at least 75%, 80%, 85%, 90% or95% sequence identity to an amino acid sequence of SEQ ID NO: 2 or SEQID NO: 3 or a peptidomimetic derivative of SEQ ID NO: 2 or SEQ ID NO: 3.The statement that the peptide is a non-native is intended to excludethe native peptides of parent proteins.

Compositions comprising a lacritin peptide or bioactive fragment orderivative thereof have use in treating ocular surface-associateddiseases, disorders, and conditions, including dry eye. Accordingly, inone embodiment lacritin polypeptide comprising compositions are used totreat such diseases, disorders, and conditions.

In accordance with one embodiment, a composition is provided comprisinga heparinase peptide, a non-native peptide, or a peptidomimeticderivative thereof, wherein the peptide comprises a sequence selectedfrom the group consisting of SEQ ID NO: 4 or a sequence that differsfrom SEQ ID NO: 4 by 1, 2, 3, 4 or 5 amino acids, and homologs andfragments thereof. In one embodiment a heparinase peptide is providedthat differs from SEQ ID NO: 4 by 1, 2, 3, 4 or 5 amino acidmodifications. In one embodiment, the amino acid modifications are aminoacid substitution, and in one embodiment the substitutions areconservative amino acid substitutions.

In some embodiments, the peptide of the present disclosure comprises anamino acid sequence which has at least 75%, 80%, 85%, 90% or 95%sequence identity to the amino acid sequence SEQ ID NO: 4 or a fragmentor homolog thereof.

In some embodiments, the peptide of the present disclosure comprises anon-native amino acid sequence which has at least 75%, 80%, 85%, 90% or95% sequence identity to an amino acid sequence of SEQ ID NO: 4 or apeptidomimetic derivative of SEQ ID NO: 4. The statement that thepeptide is a non-native is intended to exclude the native peptides ofparent proteins.

Derivatives of the peptides disclosed herein, in one embodiment, containan amino acid sequence wherein 1, 2, or 3 amino acids are deleted,substituted or added, relative to the parent peptide, as long as themodified peptide has an activity equivalent to that of peptide havingthe aforementioned amino acid sequence.

In another embodiment, a novel, isolated polypeptide having an aminoacid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:8 or a biologically active fragment, homolog, or derivative thereof isprovided. In one embodiment the polypeptide has an amino acid sequenceSEQ ID NO: 5 or a biologically active fragment, homolog, or derivativethereof. In another embodiment, the polypeptide has amino acid sequenceSEQ ID NO: 6. In another embodiment, the polypeptide has amino acidsequence SEQ ID NO: 7 or a biologically active fragment, homolog, orderivative thereof. In another embodiment, the polypeptide has aminoacid sequence SEQ ID NO: 8 or a biologically active fragment, homolog,or derivative thereof. In another embodiment, the present inventionprovides an isolated polypeptide comprising amino acid sequence SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or a biologicallyactive fragment, homolog, or derivative thereof for use in therapy. Inone embodiment, the present invention provides an purified polypeptidecomprising amino acid sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,or SEQ ID NO: 8 or a biologically active fragment, homolog, orderivative thereof for use in therapy. In one embodiment, the presentinvention provides a purified polypeptide consisting of the amino acidsequence of SEQ ID NO: 7 for use in treating an ocularsurface-associated disease, disorder, or condition.

In another embodiment, the present invention provides for the use of anisolated polypeptide comprising amino acid sequence SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or a biologically active fragment,homolog, or derivative thereof for the manufacture of a medicament forthe treatment of an ocular surface-associated disease, disorder, orcondition or any an indication recited herein. In one embodiment thepolypeptide is a purified polypeptide consisting of the amino acidsequence of SEQ ID NO: 7.

In another embodiment, the present invention provides a novelpharmaceutical composition comprising a therapeutically effective amountof at least one polypeptide comprising amino acid sequence SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or a biologically activefragment, homolog, or derivative thereof, wherein the composition issuitable for topical administration to an ocular surface of a subject.

In another embodiment, the composition comprises a polypeptide havingamino acid sequence SEQ ID NO: 5 or a biologically active fragment,homolog, or derivative thereof. In another embodiment, the compositioncomprises a polypeptide having amino acid sequence SEQ ID NO: 6 or abiologically active fragment, homolog, or derivative thereof. In anotherembodiment, the composition comprises a polypeptide having amino acidsequence SEQ ID NO: 7 or a biologically active fragment, homolog, orderivative thereof. In another embodiment, the composition comprises apolypeptide having amino acid sequence SEQ ID NO: 8 or a biologicallyactive fragment, homolog, or derivative thereof.

In one embodiment, a composition of the invention further comprises acarrier. In one aspect, the carrier is buffered saline. In one aspect, acomposition of the invention is a pharmaceutical composition. In oneaspect, a pharmaceutical composition of the invention comprises apharmaceutically-acceptable carrier. In one aspect, the carrier isbuffered saline. In one aspect, a pharmaceutical composition of theinvention further comprises at least one additional therapeutic agent.In another embodiment, the composition further comprises bufferedsaline. In another embodiment, the buffer is phosphate buffer. Inanother embodiment, the buffer is selected from sodium phosphate,disodium phosphate, potassium phosphate, dipotassium phosphate, and acombination thereof.

In another embodiment, the composition further comprises a salt selectedfrom NaCl and KCl. In another embodiment, the pH of the solution isselected from 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, and 8. Inanother embodiment, the pH is 7.4

An example of a buffered saline suitable for the present inventioncomprises H₂O and the following components.

Concentration Concentration Salt mmol/L g/L NaCl 137 8.0 KCl 2.7 0.2Na₂HPO₄ 10 1.44 KH₂PO₄ 1.8 0.24

In another embodiment, the present invention provides a novel method oftreating dry eye, comprising contacting an ocular surface of a subjectin need thereof with a composition the present invention. In accordancewith one embodiment treatment with a lacritin based compositiondisclosed herein results in one or more of the following effects:

a) the treatment restores or increases tearing;

b) the treatment restores or increases tearing without causing orincreasing inflammation;

c) the treatment restores basal tearing;

d) the treatment suppresses lacrimal gland inflammation;

e) the treatment diminishes the susceptibility of the eye to cornealstaining;

f) the treatment diminishes tear osmolarity;

g) the treatment improves ocular surface health;

h) the treatment stimulates lacrimal glands;

i) the treatment stimulates meibomian glands;

j) the treatment stimulates conjunctival goblet cells;

k) the treatment stimulates corneal sensory nerves;

l) the treatment increases the level of a shed and deglycanated 90 kDaform of syndecan-1 in the tears of the treated subject;

m) the treatment decreases the level of a 25 kDa fragment of SDC-1ectodomain in the tears of the treated subject;

n) the treatment decreases the level of inactive lacritin-C splicevariant in the tears of the treated subject;

o) the treatment increases the level of latent heparanase in the tearsof the treated subject; and

p) the treatment decreases the level of activated heparanase in thetears of the treated subject.

Patients suitable for treatment using a lacritin containing compositioninclude patients having one or more of the following conditions:

a) the tears of the subject, prior to treatment, contain low levels of90 kDa deglycanated SDC-1 compared to normal, non-dry eye tears;

b) the tears of the subject, prior to treatment, contain elevated levelsof 25 kDa SDC-1 compared to normal, non-dry eye tears;

c) the tears of the subject, prior to treatment, contain elevated levelsof inactive lacritin-C splice variant compared to normal, non-dry eyetears;

d) the tears of the subject, prior to treatment, contain low levels oflatent heparanase compared to normal, non-dry eye tears;

e) the tears of the subject, prior to treatment, contain elevated levelsof activated heparanase compared to normal, non-dry eye tears; and

f) the subject is recovering from PRK (photorefractive keratectomy) orLASIK (Laser-Assisted in situ Keratomileusis) surgery or other surgicalprocedure of the eye, and includes any subject who underwent PRK orLASIK surgery and is suffering from dry eye, regardless of the timesince receiving the surgery. In one embodiment a subject havingundergone PRK or LASIK surgery within the past day, month, six months,year, or even years can receive benefit from treatment using a lacritincontaining formulation as disclosed herein.

In accordance with one embodiment a method for identifying a subjectafflicted with insufficient tears to lubricate and nourish the eye,deriving from either insufficient tear quantity or have a poor qualityof tears. In one embodiment the method comprises screening a tear sampleobtained from said subject for the presence of at least one proteinselected from the group consisting of

-   -   latent heparanase/active heparanase;    -   90 kDa deglycanated SDC-1;    -   25 kDa SDC-1; and    -   inactive lacritin-C splice variant,        wherein the concentration of latent heparanase, or active        heparanase, and 90 kDa deglycanated SDC-1 is measured, and a        decreased level of latent heparanase, or increase in active        heparanase, (relative to levels present in tears from a normal        eye) and/or a decreased level of 90 kDa deglycanated SDC-1        (relative to levels present in tears from a normal eye) and/or        detection of 25 kDa SDC-1 in the tear sample, and/or detection        of inactive lacritin-C splice variant in the tear sample        identifies subjects having dry eye. Detection of any one of the        individual four conditions or any combination thereof indicates        a subject suffering from dry eye that would could benefit from        the topical administration of a composition comprising a        lacritin polypeptide, including for example the lacritin        fragment of SEQ ID NO: 7.

In accordance with one embodiment a tear sample is obtained from asubject and the concentration of the 90 kDa deglycanated SDC-1 and 25kDa SDC-1 are measured. Decreased levels of 90 kDa deglycanated SDC-1coupled with increased levels of 25 kDa SDC-1, relative toconcentrations of those peptides in tears from a normal eye, identifiessubjects having dry eye. In one embodiment a tear sample is obtainedfrom a subject and the sample is screened for the presence of 25 kDaSDC-1, wherein detection of 25 kDa SDC-1 identifies a subject having dryeye. In one embodiment a tear sample is obtained from a subject and thesample is screened for the presence of inactive lacritin-C splicevariant, wherein detection of inactive lacritin-C splice variantidentifies a subject having dry eye. In one embodiment a tear sample isobtained from a subject and the sample is screened for the presence of25 kDa SDC-1 and inactive lacritin-C splice variant, wherein detectionof 25 kDa SDC-1 and inactive lacritin-C splice variant identifies asubject having dry eye.

Advantageously, these markers of dry eye can serve as a basis foridentifying subjects who will benefit from lacritin therapy.Accordingly, in one embodiment a method is provided for treating dry eyewherein the first step involves identifying those subjects suitable fortreatment. In one embodiment the method of treating a subject for dryeye comprises obtaining a tear sample from the subject, and detectingthe presence of at least one protein selected from the groups consistingof latent heparanase/active heparanase, 90 kDa deglycanated SDC-1, 25kDa SDC-1, and inactive lacritin-C splice variant, wherein a decreasedlevel of latent heparanase, or increase in active heparanase, (relativeto levels present in tears from a normal eye), a decreased level of 90kDa deglycanated SDC-1 (relative to tears from a normal eye), detectionof 25 kDa SDC-1, and/or detection of inactive lacritin-C splice variantidentifies subjects having dry eye. Those subjects identified as havingdry eye based on detected levels of latent heparanase, activeheparanase, 90 kDa deglycanated SDC-1, 25 kDa SDC-1, and/or inactivelacritin-C splice variant are then administered a composition comprisinga lacritin polypeptide, including for example the peptide of SEQ ID NO:7. More particularly, the ocular surface of the subject is contactedwith a pharmaceutical composition comprising lacritin or a bioactivefragment thereof.

In accordance with one embodiment a subject identified as afflicted withdry eye is contacted with a bioactive fragment of lacritin selected fromthe group consisting of

KQFIENGSEFAQKLLKKFS (SEQ ID NO: 5);

KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7);

KQFIENGSEFANKLLKKFS (SEQ ID NO: 6); and

KQFIENGSEFANKLLKKFSLLKPWA (SEQ ID NO: 8) or a derivative thereof thatdiffers from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 byone or two amino acid substitutions. In one embodiment the bioactivefragment of lacritin consists of KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO:7).

In accordance with one embodiment a subject afflicted with dry eye thatis treatable with a lacritin therapy is identified by testing the tearsof the subject for the presence of lacritin monomer, wherein a selectivedecrease in monomer compared to a control indicates for dry eye. Thecontrol is a subject (or group of subjects) not suffering from dry eye.In another embodiment a subject afflicted with dry eye that is treatablewith a lacritin therapy is identified by testing the tears of thesubject for the presence of 90 kDa deglycanated SDC-1, wherein thedetection of normal levels of 90 kDa deglycanated SDC-1 does notindicate for dry eye. In another embodiment a subject afflicted with dryeye that is treatable with a lacritin therapy is identified bycollecting the tears of the subject and separating the proteins of thetears via their weight. In one embodiment the proteins of tears areseparated through the use of SDS-PAGE.

In another embodiment, testing is performed by contacting the tears ofthe subject with a test strip, comprising an agent capable of detectingthe presence of 25 kDa SDC-1. In one embodiment the agent is anantibody, optionally a monoclonal antibody.

In another embodiment, testing is performed by contacting the tears ofthe subject with a test strip, comprising an agent capable of detectingthe presence of 90 kDa deglycanated SDC-1. In one embodiment the agentis an antibody, optionally a monoclonal antibody.

In another embodiment, the tears are tested for the presence of 25 kDaSDC-1 and 90 kDa deglycanated SDC-1, wherein the presence of normallevels of 90 kDa deglycanated SDC-1 does not indicate for dry eye andthe presence of 25 kDa SDC-1 indicates for dry eye. In anotherembodiment, the method of identifying patients afflicted with dry eyefurther comprises a step of treating the subject with a composition ofthe present invention. In one embodiment an ocular surface of a subjectfound to have 25 kDa SDC-1 in their tears is treated with a lacritin, orlacritin fragment, containing composition.

In another embodiment, the present invention provides a novel method ofidentifying a subject having dry eye, comprising testing the tears ofthe subject for the presence of 25 kDa SDC-1, wherein the presence of 25kDa SDC-1 indicates for dry eye. In one embodiment, testing is performedby collecting the tears of the subject and separating the proteins ofthe tears based on their weight. In one embodiment the separation of thepeptides is performed using SDS-PAGE.

In another embodiment, a novel method of identifying a subject havingdry eye is provided wherein the tears of the subject are tested for thepresence of inactive lacritin-C splice variant, wherein the presence ofinactive lacritin-C splice variant indicates for dry eye. In oneembodiment the presence of inactive lacritin-C splice variant isdetected by collecting the tears of the subject and separating theproteins of the tears via their weight. In one embodiment the proteinsof the tears are separated through the use of g SDS-PAGE. In anotherembodiment, testing is performed by contacting the tears of the subjectwith a test strip, comprising an agent capable of detecting the presenceof inactive lacritin-C splice variant. In one embodiment the agent is anantibody, optionally a monoclonal antibody. In a further embodiment amethod of treatment is provided comprising contacting an ocular surfaceof a subject found to have inactive lacritin-C splice variant in theirtears with a composition of the present invention.

In another embodiment, a novel method of identifying a subject havingdry eye comprises testing the tears of the subject for the presence ofactive and latent heparanase, wherein the presence of elevated activeheparanase or depressed latent heparanase indicates for dry eye. In oneembodiment the testing is performed by collecting the tears of thesubject and separating the proteins of the tears via their weight andblotting with an anti-heparanase antibody capable of detecting latentand active heparanase.

In one embodiment, a subject suffering from evaporative dry eye, cornealinflammation, or corneal ulceration is treated by contacting the cellsof a subject in need thereof with a lacritin polypeptide comprisingcomposition disclosed herein.

In another embodiment, the present invention provides a novel method ofenhancing the proliferation of human corneal epithelial cells orlacrimal acinar cells, wherein the method comprises contacting the cellsof a subject in need thereof with a composition of the presentinvention. In one embodiment the lacritin peptide compositions disclosedherein are used to enhance the proliferation of the subject's cornealepithelial cells, enhance the proliferation of the subject's lacrimalacinar cells or inhibit epithelial cell apoptosis or other forms ofepithelial cell death. In one embodiment the method comprises contactingthe cells of a subject in need thereof with a lacritin peptidecomposition disclosed herein, wherein the cells are selected from thecorneal cells, conjunctival cells, or a combination of both.

In another embodiment, the epithelial cell cells contacted with alacritin peptide have been subjected to an insult. In one embodiment thelacritin peptide consists of the sequence of SEQ ID NO: 7. In oneembodiment, the insult is selected from the group consisting ofblepharitis, dry eye, conjunctivitis, Sjogren's syndrome, cornealabrasion, ulceration, bacterial infection, direct trauma, surgery,radiant energy, ionizing energy, viral infection, fungal infection,parasitic infection, keratitis, systemic dermatologic disorders,collagen vascular diseases, Reiter's disease, and Behcet's disease.

In another embodiment a method of treating the diseases of lysosomalclearing is provided wherein the method comprises contacting an ocularsurface of a subject with a composition of the present disclosure. Inone embodiment, the diseases are selected from glaucoma and age-relatedmacular degeneration (AMD). In one embodiment the method comprisescontacting an ocular surface of a subject with a composition comprisinga lacritin peptide, wherein the peptide consists of the sequence of SEQID NO: 7.

In another embodiment, the present invention relates to the treatment ofdiseases of lysosomal clearing. Such diseases include: glaucoma,age-related macular and degeneration (AMD). While not wishing to bebound by scientific theory, it is believed that lacritin triggers theautophagic capture and lysosomic degradation of intracellular aggregated(toxic) proteins in stressed cells. In AMD, the buildup of ‘drusen’ isboth intracellular in retinal pigment epithelial cells and extracellularnearby these cells. It is expected that if a topical administration of apolypeptide of the present invention (e.g., lacritin or polypeptide ofSEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8) infiltratesdeep into the eye it will stimulate RPE autophagy to deplete drusen. Inopen angle glaucoma, stress in the trabecular meshwork cells leads tobuild up of intracellular material that accumulates. Although autophagyis chronically elevated, this is unhealthy for cells. Instead, lacritinforces a rapid and transient bolus of accelerated autophagy sufficientto clear offending accumulating protein. Autophagy then returns tobaseline. A polypeptide of the present disclosure is expected to gainaccess to these cells.

In another embodiment, the present invention provides a novelbactericidal composition. In accordance with one embodiment abactericidal composition is provided comprising a C-terminal fragment oflacritin selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQID NO: 8 or a derivative thereof that differs from SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 by 1, 2, or 3 amino acidsubstitutions. In one embodiment the composition is suitable for topicaladministration to an ocular surface of a subject. In one embodiment thelacritin derivative that differs from SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7 or SEQ ID NO: 8 by 1, 2 or 3 amino acid substitutions, has aminoacid substitutions located at positions selected from positions 4, 6, 8,10, 17 and 19 relative to the numbering of SEQ ID NO: 7. These positionsshow variability among the highly conserved C-terminal regions ofprimate species (see FIG. 9). In one embodiment the lacritin derivativediffers from SEQ ID NO: 7 or SEQ ID NO: 8 by 1 or 2 amino acidsubstitutions at positions 4 and/or 19 relative to the numbering of SEQID NO: 7. In one embodiment the amino acid substitutions areconservative amino acid substitutions. In accordance with one embodimenta bactericidal composition is provided comprising a C-terminal fragmentof lacritin selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, orSEQ ID NO: 8. In accordance with one embodiment a bactericidalcomposition is provided comprising a C-terminal fragment of lacritinselected from SEQ ID NO: 7, or SEQ ID NO: 8, and in one embodiment theC-terminal fragment of lacritin consists of KQFIENGSEFAQKLLKKFSLLKPWA(SEQ ID NO: 7).

In one embodiment a bactericidal composition is provided comprising afirst anti-bacterial agent, which is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:8 or a biologically active fragment, homolog, or derivative thereof,wherein the composition is suitable for topical administration to anocular surface of a subject. In one aspect, the polypeptide has aminoacid sequence SEQ ID NO: 5. In another aspect, the polypeptide has aminoacid sequence SEQ ID NO: 6. In another aspect, the polypeptide has aminoacid sequence SEQ ID NO: 7. In another aspect, the polypeptide has aminoacid sequence SEQ ID NO: 8. In one aspect, the bactericidal compositionfurther comprises a bactericidally-acceptable carrier.

In one embodiment the bactericidal composition further comprises asecond anti-bacterial agent. In one embodiment, the composition furthercomprises an anti-microbial agent. Suitable ophthalmic anti-microbialagents are known to those skilled in the art and include those describedin U.S. Pat. Nos. 5,300,296, 6,316,669, 6,365,636 and 6,592,907, thedisclosures of which are incorporated herein. Examples of anti-microbialagents suitable for use in accordance with the present invention includebenzalkonium chloride, benzethonium chloride, benzyl alcohol,chlorobutanol, chlorhexidine digluconate or diacetate, methyl and propylhydroxybenzoate (parabens), phenylethyl alcohol, phenylmercuric acetateor nitrate, sorbic acid, and thimerosal.

In one embodiment a second anti-bacterial agent is present in thebactericidal composition and in one embodiment the second anti-bacterialagent is one that is naturally present in mammalian eyes. In oneembodiment the second anti-bacterial agent is lysozyme. In accordancewith one embodiment the bactericidal composition comprises a C-terminalfragment of lacritin and a second anti-bacterial agent, wherein theratio of C-terminal fragment of lacritin and a second anti-bacterialagent is at least 2:1. In one embodiment the bactericidal compositioncomprises a C-terminal fragment of lacritin and lysozyme, wherein theweight ratio of lysozyme to the C-terminal fragment of lacritin is from4:1 to 3:1. In one embodiment the C-terminal fragment of lacritinconsists of KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7).

In accordance with one embodiment, a method is provided for treatinginfections of the eye. The method comprises the step of topicallyadministering a composition comprising a lacritin polypeptide to theeye. In another embodiment, the present invention provides a novelmethod of treating a corneal infection, comprising contacting the corneaof a subject in need thereof with a bactericidal composition of thepresent invention. In one embodiment a peptide consisting of thesequence of the KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7) is used in themanufacture of a medicament for the treatment of dry eye or treatment ofa corneal infection.

In another embodiment, a method of treating a subject suffering from dryeye by contacting an ocular surface of a subject found to have activeheparanase in their tears with a composition of the present invention.

In another embodiment, the present invention provides a novel containercomprising a composition of the present invention, wherein thecomposition is in the form of an eye drop and in a volume sufficient for1 dosage. In another embodiment, the composition is in the form of aneye drop and in a volume sufficient for 1-2 dosages. In anotherembodiment, the composition is in the form of an eye drop and in avolume sufficient for up to 1 week, 2 weeks, 3 weeks, or 4 weeks. Inanother embodiment, the container is in the form of a single use ampule,a bottle formed to dispense drops of the composition, or a bottlecomprising: a body and a cap, wherein an eye dropper connect to the capor part of the cap.

The present invention can also be practiced using methods described inU.S. Pat. Nos. 7,648,964, 7,459,440, 7,320,870, and 7,932,227, andpublications WO 98/27205 (Jacobs et al., published Jun. 25, 1998),Sanghi et al., 2001, J. Mol. Biol., 310:127, Wang et al., 2006, J. CellBiol., 174(5):689-700, Epub 2006 Aug. 21, Ma et al., J. Cell Biol.,2006, 174:7:1097-1106, Zhang et al., J. Biol. Chem., 2013,288(17):12090-101: Epub 2013 Mar. 15, the contents of which areincorporated by reference in their entirety herein.

Various aspects and embodiments of the invention are described infurther detail below.

In accordance with one embodiment a novel mechanism for the molecularidentification of dry eye disease is coupled with a restorative therapythat addresses cause. In one embodiment the method of identifying dryeye relates to the discovery that a ˜90 KDa deglycanated form ofsyndecan-1 is abundant in tears of normal individuals but notindividuals suffering from dry eye, whereas a ˜25 kDa syndecan-1fragment is detectable in dry, but not normal tears. Also disclosedherein is the discovery that topical lacritin, the agonist ofdeglycanated syndecan-1, sensitizes corneal sensory nerves to drying ofthe surface of the eye and increases the neural wet response.Accordingly, one embodiment of the present invention is directed to amethod of identifying dry eye by detecting abnormally low levels of −90kDa and/or the presence of 25 kDa syndecan-1 in tears. Anotherembodiment is directed to a method of increasing the corneal neural dryand wet responses by administering topical lacritin or lacritinfragments, synthetic peptides or mimetics.

Current tear supplements are not popular with subjects, in part becausethe relief obtained from such products is very brief (less than 15 min).Examples of the tear substitution approach include the use of buffered,isotonic saline solutions, aqueous solutions containing water solublepolymers that render the solutions more viscous and thus less easilyshed by the eye. Tear reconstitution is also attempted by providing oneor more components of the tear film such as phospholipids and oils.Examples of these treatment approaches are disclosed in U.S. Pat. No.4,131,651 (Shah et al.), U.S. Pat. No. 4,370,325 (Packman), U.S. Pat.No. 4,409,205 (Shively), U.S. Pat. Nos. 4,744,980 and 4,883,658 (Holly),U.S. Pat. No. 4,914,088 (Glonek), U.S. Pat. No. 5,075,104 (Gressel etal.) and U.S. Pat. No. 5,294,607 (Glonek et al.) the disclosures ofwhich are incorporated herein. Existing ophthalmic formulations may alsoinclude TGF-beta, corticosteroids, or androgens. All are non-specificfor the eye and have systemic effects. In contrast, lacritin is highlyrestricted to the eye and is a natural constituent of human tears andthe tear film.

An ophthalmic formulation comprising lacritin, or fragments, homologs,or derivatives thereof (for example, an artificial tear fluidscontaining lacritin), is highly desirable due to the activity oflacritin and its localized effects. In accordance with one embodiment ofthe invention, compositions comprising lacritin, or bioactive fragmentsthereof, are used to enhance corneal wound healing, and/or treatsubjects having deficient tear output. The lacritin compositions of thepresent invention can be formulated using standard ophthalmiccomponents, and preferably, the compositions are formulated assolutions, suspensions, and other dosage forms for topicaladministration. Aqueous solutions are generally preferred, based on easeof formulation, biological compatibility (especially in view of themalady to be treated, e.g., dry eye-type diseases and disorders), aswell as a subject's ability to easily administer such compositions bymeans of instilling one to two drops of the solutions in the affectedeyes. However, the compositions may also be suspensions, viscous orsemi-viscous gels, or other types of solid or semi-solid compositions.

The compositions of the present invention may include surfactants,preservative agents, antioxidants, tonicity agents, buffers,preservatives, co-solvents and viscosity building agents. Varioussurfactants useful in topical ophthalmic formulations may be employed inthe present compositions. These surfactants may aid in preventingchemical degradation of the lacritin polypeptide and also prevent thelacritin polypeptide from binding to the containers in which thecompositions are packaged. Examples of surfactants include, but are notlimited to: Cremophor™ EL, polyoxyl 20 ceto stearyl ether, polyoxyl 40hydrogenated castor oil, polyoxyl 23 lauryl ether and poloxamer 407 maybe used in the compositions. Antioxidants may be added to compositionsof the present invention to protect the lacritin polypeptide fromoxidation during storage. Examples of such antioxidants include, but arenot limited to, vitamin E and analogs thereof, ascorbic acid andderivatives, and butylated hydroxyanisole (BHA).

Existing artificial tears formulations can also be used aspharmaceutically acceptable carriers for the lacritin active agent. Thusin one embodiment, a lacritin polypeptide is used to improve existingartificial tear products for Dry Eye syndromes, as well as developproducts to aid corneal wound healing. Examples of artificial tearscompositions useful as carriers include, but are not limited to,commercial products, such as Tears Naturale™, Tears Naturale II™, TearsNaturale Free™, and Bion Tears™. (Alcon Laboratories, Inc., Fort Worth,Tex.). Examples of other phospholipid carrier formulations include thosedisclosed in U.S. Pat. No. 4,804,539 (Guo et al.), U.S. Pat. No.4,883,658 (Holly), U.S. Pat. No. 4,914,088 (Glonek), U.S. Pat. No.5,075,104 (Gressel et al.), U.S. Pat. No. 5,278,151 (Korb et al.), U.S.Pat. No. 5,294,607 (Glonek et al.), U.S. Pat. No. 5,371,108 (Korb etal.), U.S. Pat. No. 5,578,586 (Glonek et al.); the foregoing patents areincorporated herein by reference to the extent they disclosephospholipid compositions useful as phospholipid carriers of the presentinvention. In accordance with one embodiment an topical ophthalmicformulation is provided comprising a lacritin peptide consisting of thesequence of SEQ ID NO: 7 and a pharmaceutically acceptable carrier. Inone embodiment the composition further comprises a phospholipid. In analternative embodiment the composition further comprises a surfactant,preservative agent, antioxidant, tonicity agent, buffer, preservative,co-solvent and/or viscosity building agents.

Other compounds may also be added to the ophthalmic compositions of thepresent disclosure to increase the viscosity of the carrier. Examples ofviscosity enhancing agents include, but are not limited to:polysaccharides, such as hyaluronic acid and its salts, chondroitinsulfate and its salts, dextrans, various polymers of the cellulosefamily; vinyl polymers; and acrylic acid polymers. In general, thephospholipid carrier or artificial tears carrier compositions willexhibit a viscosity of 1 to 400 centipoises (“cps”). Preferredcompositions containing artificial tears or phospholipid carriers andwill exhibit a viscosity of about 25 cps.

Topical ophthalmic products are typically packaged in multidose form.Preservatives are thus required to prevent microbial contaminationduring use. Suitable preservatives include: benzalkonium chloride,chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben,phenylethyl alcohol, edetate disodium, sorbic acid, polyquarternium-1,or other agents known to those skilled in the art. Such preservativesare typically employed at a level of from 0.001 to 1.0% w/v. Unit dosecompositions of the present invention will be sterile, but typicallyunpreserved. Such compositions, therefore, generally will not containpreservatives.

Because the gene promoter regulating lacritin gene expression is themost specific of any previously described lacrimal gland gene, theregulatory elements of this gene could be used to express other geneproducts in the eye. In particular, the lacritin gene promoter can beoperably linked to a wide variety of exogenous genes to regulate theexpression of the gene products to the lacrimal gland and/or used asgene therapy to treat Dry Eye syndromes.

The peptides of the present disclosure may be readily prepared bystandard, well-established techniques, such as solid-phase peptidesynthesis (SPPS) as described by Stewart et al. in Solid Phase PeptideSynthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill.;and as described by Bodanszky and Bodanszky in The Practice of PeptideSynthesis, 1984, Springer-Verlag, New York. At the outset, a suitablyprotected amino acid residue is attached through its carboxyl group to aderivatized, insoluble polymeric support, such as cross-linkedpolystyrene or polyamide resin. “Suitably protected” refers to thepresence of protecting groups on both the α-amino group of the aminoacid, and on any side chain functional groups. Side chain protectinggroups are generally stable to the solvents, reagents and reactionconditions used throughout the synthesis, and are removable underconditions which will not affect the final peptide product. Stepwisesynthesis of the oligopeptide is carried out by the removal of theN-protecting group from the initial amino acid, and couple thereto ofthe carboxyl end of the next amino acid in the sequence of the desiredpeptide. This amino acid is also suitably protected. The carboxyl of theincoming amino acid can be activated to react with the N-terminus of thesupport-bound amino acid by formation into a reactive group such asformation into a carbodiimide, a symmetric acid anhydride or an “activeester” group such as hydroxybenzotriazole or pentafluorophenly esters.

Examples of solid phase peptide synthesis methods include the BOC methodwhich utilized tert-butyloxcarbonyl as the α-amino protecting group, andthe FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protectthe α-amino of the amino acid residues, both methods of which are wellknown by those of skill in the art.

Incorporation of N- and/or C-blocking groups can also be achieved usingprotocols conventional to solid phase peptide synthesis methods. Forincorporation of C-terminal blocking groups, for example, synthesis ofthe desired peptide is typically performed using, as solid phase, asupporting resin that has been chemically modified so that cleavage fromthe resin results in a peptide having the desired C-terminal blockinggroup. To provide peptides in which the C-terminus bears a primary aminoblocking group, for instance, synthesis is performed using ap-methylbenzhydrylamine (MBHA) resin so that, when peptide synthesis iscompleted, treatment with hydrofluoric acid releases the desiredC-terminally amidated peptide. Similarly, incorporation of anN-methylamine blocking group at the C-terminus is achieved usingN-methylaminoethyl-derivatized DVB, resin, which upon HF treatmentreleases a peptide bearing an N-methylamidated C-terminus. Blockage ofthe C-terminus by esterification can also be achieved using conventionalprocedures. This entails use of resin/blocking group combination thatpermits release of side-chain peptide from the resin, to allow forsubsequent reaction with the desired alcohol, to form the esterfunction. FMOC protecting group, in combination with DVB resinderivatized with methoxyalkoxybenzyl alcohol or equivalent linker, canbe used for this purpose, with cleavage from the support being effectedby TFA in dicholoromethane. Esterification of the suitably activatedcarboxyl function e.g. with DCC, can then proceed by addition of thedesired alcohol, followed by deprotection and isolation of theesterified peptide product.

Incorporation of N-terminal blocking groups can be achieved while thesynthesized peptide is still attached to the resin, for instance bytreatment with a suitable anhydride and nitrile. To incorporate anacetyl-blocking group at the N-terminus, for instance, the resin-coupledpeptide can be treated with 20% acetic anhydride in acetonitrile. TheN-blocked peptide product can then be cleaved from the resin,deprotected and subsequently isolated.

To ensure that the peptide obtained from either chemical or biologicalsynthetic techniques is the desired peptide, analysis of the peptidecomposition should be conducted. Such amino acid composition analysismay be conducted using high-resolution mass spectrometry to determinethe molecular weight of the peptide. Alternatively, or additionally, theamino acid content of the peptide can be confirmed by hydrolyzing thepeptide in aqueous acid, and separating, identifying and quantifying thecomponents of the mixture using HPLC, or an amino acid analyzer. Proteinsequenators, which sequentially degrade the peptide and identify theamino acids in order, may also be used to determine definitely thesequence of the peptide.

Prior to its use, the peptide is purified to remove contaminants. Inthis regard, it will be appreciated that the peptide will be purified tomeet the standards set out by the appropriate regulatory agencies. Anyone of a number of a conventional purification procedures may be used toattain the required level of purity including, for example,reversed-phase high-pressure liquid chromatography (HPLC) using analkylated silica column such as C4-, C8- or C18-silica. A gradientmobile phase of increasing organic content is generally used to achievepurification, for example, acetonitrile in an aqueous buffer, usuallycontaining a small amount of trifluoroacetic acid. Ion-exchangechromatography can be also used to separate peptides based on theircharge.

It will be appreciated, of course, that the peptides or antibodies,derivatives, or fragments thereof may incorporate amino acid residueswhich are modified without affecting activity. For example, the terminimay be derivatized to include blocking groups, i.e. chemicalsubstituents suitable to protect and/or stabilize the N- and C-terminifrom “undesirable degradation”, a term meant to encompass any type ofenzymatic, chemical or biochemical breakdown of the compound at itstermini which is likely to affect the function of the compound, i.e.sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C1-05 branchedor unbranched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm) group. Desamino analogs of amino acids are alsouseful N-terminal blocking groups, and can either be coupled to theN-terminus of the peptide or used in place of the N-terminal reside.Suitable C-terminal blocking groups, in which the carboxyl group of theC-terminus is either incorporated or not, include esters, ketones oramides. Ester or ketone-forming alkyl groups, particularly lower alkylgroups such as methyl, ethyl and propyl, and amide-forming amino groupssuch as primary amines (—NH2), and mono- and di-alkylamino groups suchas methylamino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like are examples of C-terminal blockinggroups. Descarboxylated amino acid analogues such as agmatine are alsouseful C-terminal blocking groups and can be either coupled to thepeptide's C-terminal residue or used in place of it. Further, it will beappreciated that the free amino and carboxyl groups at the termini canbe removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without effect on peptide activity.

Other modifications can also be incorporated without adversely affectingthe activity and these include, but are not limited to, substitution ofone or more of the amino acids in the natural L-isomeric form with aminoacids in the D-isomeric form. Thus, the peptide may include one or moreD-amino acid resides, or may comprise amino acids which are all in theD-form. Retro-inverso forms of peptides in accordance with the presentinvention are also contemplated, for example, inverted peptides in whichall amino acids are substituted with D-amino acid forms.

Acid addition salts of the present invention are also contemplated asfunctional equivalents. Thus, a peptide in accordance with the presentinvention treated with an inorganic acid such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organicacid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic,malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie,mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclicand the like, to provide a water soluble salt of the peptide is suitablefor use in the invention.

The present disclosure also provides for analogs of proteins. Analogscan differ from naturally occurring proteins or peptides by conservativeamino acid sequence differences or by modifications which do not affectsequence, or by both.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the protein or peptide, do notnormally alter its function. To that end, 10 or more conservative aminoacid changes typically have no effect on peptide function. In accordancewith one embodiment conservative amino acid substitutions can includesubstitutions within the following groups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine;

phenylalanine, tyrosine.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

Also included are polypeptides or antibody fragments which have beenmodified using ordinary molecular biological techniques so as to improvetheir resistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring synthetic amino acids. The peptides of theinvention are not limited to products of any of the specific exemplaryprocesses listed herein.

The skilled artisan will be aware that, in general, amino acidsubstitutions in a peptide typically involve the replacement of an aminoacid with another amino acid of relatively similar properties (i.e.,conservative amino acid substitutions). The properties of the variousamino acids and effect of amino acid substitution on protein structureand function have been the subject of extensive study and knowledge inthe art.

For example, one can make the following isosteric and/or conservativeamino acid changes in the parent polypeptide sequence with theexpectation that the resulting polypeptides would have a similar orimproved profile of the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: includingalanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid,S-cyclohexylalanine or other simple alpha-amino acids substituted by analiphatic side chain from C1-10 carbons including branched, cyclic andstraight chain alkyl, alkenyl or alkynyl substitutions.

Substitution of aromatic-substituted hydrophobic amino acids: includingphenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine,2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine,histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro,chloro, bromo, or iodo) or alkoxy-substituted forms of the previouslisted aromatic amino acids, illustrative examples of which are: 2-, 3-or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2,3, or 4-biphenylalanine, 2′, -3′, - or4′-methyl-2, 3 or 4-biphenylalanine, and 2- or 3-pyridylalanine.

Substitution of amino acids containing basic functions: includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, alkyl, alkenyl, or aryl-substituted (from C1-C10 branched,linear, or cyclic) derivatives of the previous amino acids, whether thesubstituent is on the heteroatoms (such as the alpha nitrogen, or thedistal nitrogen or nitrogens, or on the alpha carbon, in the pro-Rposition for example. Compounds that serve as illustrative examplesinclude: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine,3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma′-diethyl-homoarginine.Included also are compounds such as alpha methyl arginine, alpha methyl2,3-diaminopropionic acid, alpha methyl histidine, alpha methylornithine where alkyl group occupies the pro-R position of the alphacarbon. Also included are the amides formed from alkyl, aromatic,heteroaromatic (where the heteroaromatic group has one or morenitrogens, oxygens, or sulfur atoms singly or in combination) carboxylicacids or any of the many well-known activated derivatives such as acidchlorides, active esters, active azolides and related derivatives) andlysine, ornithine, or 2,3-diaminopropionic acid.

Substitution of acidic amino acids: including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids.

Substitution of side chain amide residues: including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine.

Substitution of hydroxyl containing amino acids: including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine. It is also understoodthat the amino acids within each of the categories listed above can besubstituted for another of the same group.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within +/−2 is preferred, within +/−1 aremore preferred, and within +/−0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL Rockefeller University website). Forsolvent exposed residues, conservative substitutions would include: Aspand Asn; Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala andPro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg;Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr. Various matriceshave been constructed to assist in selection of amino acidsubstitutions, such as the PAM250 scoring matrix, Dayhoff matrix,Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix,Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix andRisler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded peptide sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Substantially pure protein obtained as described herein may be purifiedby following known procedures for protein purification, wherein animmunological, enzymatic or other assay is used to monitor purificationat each stage in the procedure. Protein purification methods are wellknown in the art, and are described, for example in Deutscher et al.(ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich,San Diego).

The invention also includes a kit comprising the composition of theinvention and an instructional material which describes administeringthe composition to a subject. In another embodiment, this kit comprisesa (preferably sterile) solvent suitable for dissolving or suspending thecomposition of the invention prior to administering the composition.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,and to birds including commercially relevant birds such as chickens,ducks, geese, and turkeys.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, intravenous, topical, pulmonary,intranasal, buccal, ophthalmic, intrathecal or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi liquid preparations such as liniments,lotions, oil in water or water in oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1 1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference. Typically, dosages of the compound ofthe invention which may be administered to a subject, preferably ahuman, range in amount from 1 μg to about 100 g per kilogram of bodyweight of the subject. While the precise dosage administered will varydepending upon any number of factors, including but not limited to, thetype of subject and type of disease state being treated, the age of thesubject and the route of administration.

The invention further provides for identifying a subject with dry eye.For example, proteins or peptides found in tears can be detected usingvarious methods, included, but not limited to, ELISA, immunoassay,immunofluorescence, immunohistochemistry, immunoprecipitation, andwestern blot,

In one embodiment a kit is provided comprising the composition of theinvention and an instructional material which describes administeringthe composition to a cell or a tissue of a subject. In anotherembodiment, this kit comprises a (preferably sterile) solvent suitablefor dissolving or suspending the composition of the invention prior toadministering the compound to the subject. As used herein, an“instructional material” includes a publication, a recording, a diagram,or any other medium of expression which can be used to communicate theusefulness of the peptide of the invention in the kit for effectingalleviation of the various diseases or disorders recited herein. In oneembodiment the kit provides standard curves providing informationregarding the concentration of various peptides in a normal healthy eye.Optionally, or alternately, the instructional material may describe oneor more methods of alleviation the diseases or disorders in a cell or atissue of a subject. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains thepeptide of the invention or be shipped together with a container whichcontains the peptide. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

Example 1

Identification of Dry Eye Disease

Procedures

Cell Culture, Constructs, and Antibodies

Human corneal epithelial (HCE-T) cells were purchased from the RIKENBioResource Center (Tsukuba-shi, Japan) and used between passages 3 and15. HCE-T cells were cultured and maintained in DMEM/F-12 containing 4mg/ml insulin, 100 μg/ml EGF, 500 μg/ml cholera toxin, and 5 μl/ml DMSO.Primary human corneal epithelial cells (PCS-700-010) were purchased fromATCC (Manassas, Va.) and expanded in the suggested medium.

N-terminal deletions of 45, 65, and 71 amino acids and point mutantsV69S, I73S, I98S, F104S, L108S, L109S, F112S, I68S/I73S, V91S/L109S, andL108S/L109S/F112S were developed from pLAC. All constructs wereconfirmed by DNA sequencing. Lacritin and deletion or point mutants,including deletion mutant “C-25”, were generated in Escherichia coli andpurified as described previously (Wang et al, (2006) J. Cell Biol. 174,689-700)) with additional purification over DEAE in PBS in whichlacritin is collected in the flow-through. Purified lacritin wasfilter-sterilized and stored lyophilized

Polyclonal N and C terminus-specific anti-lacritin antibodies wererespectively generated in New Zealand White rabbits against keyholelimpet hemocyanin-conjugated EDASSDSTGADPAQEAGTS (“Pep Lac N-Term”) as“anti-Pep Lac N-term” and against lacritin deletion mutant N-65 as“anti-N-65 Lac C-term” (Bio-Synthesis Inc., Lewisville, Tex.) andcharacterized. Monoclonal N terminus-specific anti-lacritin antibodieswere generated (University of Virginia Lymphocyte Culture Center) inmice against keyhole limpet hemocyanin-conjugated DPAQEAGTSKPNEEIS andscreened through three rounds of cloning against the lacritin deletionmutant C-59 as 1F5-C9-F4 (“1F5”; IgG1).

Tears and Viability Analyses

Tears were collected from 0.5% proparacaine-anesthetized eyes from atotal of normal or dry eye individuals by insertion of a filter wicking“Schirmer” strip with millimeter gradations between the lid and eye andindividually stored at −70° C. Prior to elution, the total normal or dryeye tear volume was estimated from millimeters of tears drawn into eachstrip. This defined the final volume of PBS respectively used forelution. Pooled normal or pooled dry eye tears were stored at −70° C.until use.

For FOXO3 translocation assays, HCE-T cells were grown in triplicate tosubconfluence (˜50%) on coverslips in α-MEM (5.54 mm glucose),sensitized overnight in IFNG (100 units/ml; Roche Applied Science), andtreated for 15 min with normal or dry eye tears diluted 1:100 in α-MEMtogether with TNF (50 ng/ml; PeproTech, Rocky Hill, N.J.) without orwith 10 nm lacritin or C-25. Cells were washed, fixed with 4%paraformaldehyde, and immunostained for FOXO3 (1:200; Millipore,Billerica, Mass.) followed by goat anti-rabbit secondary antibody andvisualization on a Zeiss LSM 700 microscope.

Some experiments were performed with normal tears that had beenimmunodepleted of lacritin. For immunodepletion, rabbit anti-Pep LacN-term and anti-N-65 Lac C-term were jointly immobilized on protein Abeads and washed. A rabbit preimmune column was similarly prepared formock-depleted tears. The flow-through from overnight incubation of eachwith normal tears was collected and assayed in triplicate onIFNG-sensitized cells with TNF as described above. For validation, theacid eluant from each column was separated by SDS-PAGE, transferred tonitrocellulose, and blotted for lacritin using mouse anti-lacritinantibody 1F5 and a mouse-specific, peroxidase-labeled secondary antibodyfollowed by chemiluminescence detection.

Viability was monitored using the3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) reductionassay (Invitrogen) or a Nucleocounter (New Brunswick Scientific, Edison,N.J.). Cells were seeded overnight in 24-well plates at a density of 500cells/mm2 to give rise to ˜80% confluence the next day. Then cells weresensitized overnight in IFNG (100 units/ml) in α-MEM and treated intriplicate for 15 min with 10 nm lacritin or lacritin deletion or pointmutants or with different lacritin doses in α-MEM together with TNF asdescribed above.

Inclusion of inhibitors was simultaneous with the addition of lacritinor C-25 in all viability and other experiments except where otherwisenoted. Inhibitors included PI103 (0.5 μm; EMD, Darmstadt, Germany),rapamycin (10 and 100 nm; EMD), and cyclosporin A (0.1 μm; EMD). Oneexception was 4-methylumbelliferyl-β-d-xylopyranoside (“xyloside”; 70and 80 nm; Sigma), which was added during IFNG sensitization and duringtreatment with TNF and lacritin. The assay was completed by addition ofMTT (5 mg/ml) to each well (at 37° C. for 4 h) followed by isopropanolwith 0.04 n HCl and measurement at 570 nm using a reference wavelengthof 630 nm. Viability was assayed in a Nucleocounter (New BrunswickScientific).

Results

Tears accumulate on the avascular corneal epithelium, and vascularizedconjunctiva, as a translucent film rich in proteins, lipids andmetabolites. Beyond its capacity to lubricate the lid, tears areessential for the refraction of light. Equally important andirreplaceable by drugs or drops is the role of tears in promotingcorneal epithelial health. When tears are chronically insufficient theepithelium becomes stressed and releases inflammatory cytokines thatfurther exacerbate the situation. Dry eye affects 5-6% of the generalpopulation, rising to 6-9.8% and as high as 34%, respectively inpostmenopausal women and the elderly. Although the most common eyedisease, there is no single gold standard diagnostic test, nor effectivetreatment. Current approaches include: a) subject questionnaires, b)rose bengal or lissamine green staining of ocular surface damage, c)Schirmer strip measurement of tear volume, d) tear break up time, e)tear evaporation rate, f) tear meniscus height or radius, g) tear filmindex or turnover rate, h) tear osmolarity, i) lysozyme or lactoferrinassay, and j) tear ferning analysis, each of which have numerousshortcomings.

The tear proteome is estimated to comprise 1,543 proteins, with overhalf designated as ‘intracellular’ by Gene Ontology, implying that celldeath from normal epithelial renewal may be a contributor. The onlygrowth factor-like molecule downregulated in mild to severe aqueousdeficiency was lacritin. Comparison of tears from 73 normals to 129individuals suffering from aqueous deficient dry eye by 2-D SDS PAGErevealed lacritin to be downregulated in 95% of aqueous deficient dryeye. Lacritin promotes basal tearing when added topically in rabbits.Another tear protein found to be downregulated in dray eye waslipocalin-1. Lipocalin-1 cleanses the ocular surface of lipids thatwould otherwise interfere with ocular surface wetting. Lacritin was themost severely downregulated protein in contact lens-related dryeye—perhaps in part because it is readily adsorbed on contact lenses. Itis also deficient in blepharitis, a common inflammation of the eyelid,associated with evaporative dry eye. However, 2-D SDS PAGE prior to massspectrometry is necessary to distinguish lacritin downregulation, amethod not practical for clinical use.

Several molecules are necessary for lacritin activity. An unusualdeglycanated form of syndecan-1 (SDC1) was discovered to be the maincell surface binding protein for lacritin by mass spectrometricsequencing of cell surface proteins bound to lacritin columns atphysiological salt. Validation was by affinity precipitation. SDC1 is awidely expressed cell surface heparan sulfate proteoglycan with acarboxy terminal end anchored in the plasma membrane with shortcytoplasmic tail, and an ectodomain substituted proximally withchondroitin sulfate chain(s) at serines 184 and 194 (human SDC1;numbering excludes the signal peptide), and distally with up to threeheparan sulfate chains (serines 15, 23, 25)—without or with a shortchondroitin sulfate chain. Lacritin's C-terminal α-helix binds a domainwithin SDC1 amino acids 1-50, with binding dependent on prior heparanasedeglycanation of heparan sulfate. SiRNA knockdown of SDC1 abrogateslacritin dependent mitogenic activity, as does depletion of heparanase(but not heparanase-2), but can be rescued by addition of exogenousheparanase or with bacterial heparitinase. The binding domain has beennarrowed to hydrophobic amino acids 20-30 that enhances lacritinC-terminal α-helicity. Binding was also dependent on substitution of S23and S25 (and possibly S15) with both heparan sulfate and chondroitinsulfate as a novel hybrid domain of hydrophobic core protein, heparanasecleaved heparan sulfate and adjacent chondroitin sulfate. Heparanase isnot widely expressed. N-terminal substitution of SDC1 with chondroitinsulfate is uncommon.

We looked for SDC1 in tears using a highly sensitive chemoluminescentapproach. Tears were collected onto Schirmer strips from 146 individualswho were then subjected to vision corrective photorefractive keratectomyor LASIK surgery, with further tear collection 1 day, 1 week, and 1month later. Tears were stored at −70° C., eluted with a tear equivalentvolume of PBS, pooled by time and whether normal (≥15 mm) vs dry eye (≤5mm) tears, and then separated by SDS-PAGE. Separated tear proteins weretransferred to nitrocellulose and blotted with anti-SDC1 mab A-38B.Secondary abs were precleared over a tear column, and ab C-term wasprecleared over C-59 lacritin truncation mutant. Normal tears wereunexpectedly enriched in the rare, heparanase deglycanated form of SDC1(FIG. 1) targeted by lacritin. Little of the deglycanated form of SDC1was apparent in dry eye tears. Deglycanated SDC1 can vary in molecularweight from ˜90 (FIG. 1) to ˜80 kDa or even ˜60 kDa-dependent on thelevel of O-glycosylation that can vary among different epithelia. Oneday after photorefractive keratectomy or LASIK surgery tear ˜90 kDa SDC1was indistinguishable between normal and dry eye—in keeping withsurgery-induced dry eye. A new ˜25 kDa SDC1 fragment became apparent inthose originally designated as dry eye (FIG. 1). Deglycanated SDC1 andtearing was restored in normal individuals 1 week and 1 month later.However, the dry eye-associated ˜25 kDa band remained (FIG. 1)throughout the assayed timeframe. Thus, ˜90 kDa syndecan in tears is amarker of normalcy. Those lacking ˜90 kDa syndecan have dry eye.Further, the ˜25 kDa form distinguishes dry eye individuals whounderwent PRK or LASIK.

Blotting for the inactive lacritin-c splice variant in tears also provedto be indicative of dry eye (FIG. 2A). Lacritin-c lacks sequence fromexons 4 and 5 encoding the C-terminus and has an additional sequence notpresent in native lacritin. Instead, an inactive novel C-terminus fromintron 3 is spliced in. We further discovered differences in tearheparanase with more latent heparanase in normal tears (with theexception of one day after photorefractive keratectomy or LASIK surgery;FIG. 3), whereas active heparanase was abundant in dry eye tears.Secretion of heparanase that has been processed from its latent 65 kDato active 58 kDa heterodimeric forms is stimulated by UTP. UTP is aproposed treatment for dry eye via a mechanism thought to involve theproduction of mucins. These observations suggest a potential linkagebetween lacritin, UTP and heparanase in ocular surface physiology.

Taken together, aqueous deficient dry eye tears are associated withdramatically less deglycanated SDC1 and latent heparanase, butsubstantially more SDC1 fragment and chronically active heparanase, aswell as inactive lacritin-c splice variant at the expense of normalactive lacritin. These conditions are appropriate for the exacerbationor initiation of dry eye that can be reversed by topically restoringlacritin.

Identification-Specific Treatment of Dry Eye Disease

Commonly used ‘artificial tears’ temporarily alleviate symptomsassociated with dry eye without addressing the cause of those symptoms.An ophthalmic formulation of the anti-inflammatory agent cyclosporine isnow in wide use. It and other anti-inflammatory agents are in clinicaltrials, but generally benefit only ˜15% of dry eye subjects. Rather thanfocusing on inflammatory sequelae, or applying drugs developed for otherorgan systems, there is benefit in considering the natural biology ofthe ocular surface and what is missing in dry eye. Downregulation oflacritin monomer, a natural tear protein that promotes basal tearingwhen added topically to normal rabbit eyes, may be an upstreaminstigator of dry eye disease. Why is there less lacritin monomer in dryeye? Lacritin monomer is cross-linked into inactive multimers by tissuetransglutaminase (TGM2) in tears. This was demonstrated byimmunodepleting all lacritin monomer, multimer and fragment from humantears. Recombinant lacritin spiked into immunodepleted tears formeddimers, trimers and tetramers after overnight incubation at 37° C. Inthe negative control without tears, a small amount of dimer formed.Crosslinking involves glutamine 106 within the lacritin mitogenic domain(amino acids 100-109) that targets syndecan-1. Cross-linked lacritinbinds syndecan-1 substantially less and is less active (FIG. 5; righttwo bars). Blotting suggests that normal human tears contain 0.6 μMTGM2, that thus appears to act as a negative regulator of monomericlacritin. Human corneal epithelial cells express both TGM1 and TGM2mRNA's. mRNA expression of both increases with hyperosmolar stress,particularly TGM1, however TGM1 has not been detected in tears. Thus,lacritin may be subjected to enhanced cross-linking and deactivation indry eye.

To define the lacritin domain necessary for regulation of homeostasis,truncation and point mutants were generated. Inactivity of the C-25truncation mutant defined a cytoprotective domain in the C-terminus oflacritin that was previously shown to be α-helical and likelyamphipathic, and accordingly ordered. The hydrophobic face ofamphipathic α-helices can mediate high affinity agonist—receptor orco-receptor interactions. To assay this possibility, hydrophobicresidues were singly, doubly, or triply mutated. Also generated weretruncations, and the C-terminus (I3′) of the lacritin-c splice variantwith completely different sequence from wild type. Amino acid numberingthroughout is of mature protein without signal peptide. Hydrophobic facemutants I98S, F104S, L108S/L109S/F112S, and F112S were significantlyless active (Wang et al, '13). Activity was unaffected by mutationsL65S, I68S/I78S, V69S, and I73S in an adjacent α-helix. Deleting 45, 65or 71 N-terminal amino acids had no effect, and 13 was inactive. L108,L109 and F112 interact with the syndecan-1 core protein sequence GAGAL.

Basal tears from normal individuals and from those diagnosed with dryeye were incubated with human corneal epithelial cells stressed with theinflammatory cytokines interferon-γ (INFG) and tumor necrosis factor(TNF)—much like their in vivo dry eye counterparts. Nuclear—cytoplasmictranslocation of the corneal transcription factor FOXO3 served as asimple readout for cellular stress, with cytoplasmic FOXO3 indicative ofrestored homeostasis. Nuclear FOXO3 largely transcribes for cell stressor death. In stressed cells treated with normal tears, FOXO3translocated to the cytoplasm). However with dry eye tears, FOXO3remained nuclear. Next, lacritin was immunodepleted from normal tears,although normal tears have other growth factors that might compensate.Also dry eye tears were spiked with lacritin. Dry eye tears are bothhyperosmolar and inflammatory cytokine-rich. Mock-depleted tearstranslocated FOXO3 to the cytoplasm, whereas FOXO3 remained nuclear incells treated with lacritin depleted tears. Dry eye tears spiked withlacritin, but not those spiked with lacritin truncation mutant C-25(lacking C-terminal 25 amino acids), translocated FOXO3 to thecytoplasm. Lacritin, but not C-25, also translocated FOXO3 in INFG/TNFstressed primary human corneal epithelial cells. Thus, lacritin is themaster protector of normal tears.

This test was repeated using a bioactive C-terminal fragment of lacritin(LACRIPEP; SEQ ID NO: 7). Cultured human corneal epithelial cells weretreated with inflammatory cytokines to induce stress as described above,and cells were treated with 10 nM of an inactive lacritin truncationmutant (C-25), lacritin or LACRIPEP. Measurements of cytoplasmicstaining in the FOXO3 assay (wherein nuclear FOXO3 staining isindicative of cell death) reveal LACRIPEP is equally active as lacritin(See FIG. 4A) in enhancing cell survival relative to the negativecontrol (C-25). Accordingly, applicants anticipate that LACRIPEP cansubstitute for lacritin for all applications.

Autoimmune regulator (Aire)-deficient [Aire^(−/−)] mice spontaneouslydevelop dry eye without need for dry chambers or scopolamine Aire^(−/−)mice were dosed three times daily for three weeks with 10 μl of 50 μg/mllacritin, or in controls with PBS. Several different assays monitoredthe consequences. A bioactive fragment of lacritin, LACRIPEP (SEQ ID NO:7), prevents loss of tearing as dry eye disease develops in Aire(−/−)dry eye mice (FIG. 4B; closed circles) relative to topicallyadministered PBS (opened circles), and reduced inflammation of thelacrimal gland. Topical lacritin reduced CD4+ T cell infiltration intolacrimal glands measured as the number of lymphocytic foci/permillimeter square area of lacrimal gland tissue (3.68±0.65 per mm sqlacritin vs. 9.7+1.5 per mm sq PBS; P=0.01), but had no apparent effecton the pattern or distribution of CD4+ T cells into either the cornealstroma (14.6±1.6 lacritin vs 12.4±2.1 PBS) or the limbus (29.6±2.5lacritin vs 34.6±2.9 PBS.

To assess ocular surface mucosal damage from dry eye, eyes of Aire(−/−)dry eye mice were topically administered lissamine green thatincreasingly stained PBS-treated eyes with time (See FIG. 4C). Incontrast, topical lacritin significantly decreased staining (−0.417±0.06lacritin vs 0.125±0.07 PBS; p=0.02. Further, lacritin diminished levelsof keratin 10 (skin epidermal marker), indicating a capacity to blockcorneal keratinization associated with chronic inflammation, whereaskeratin 12) expression (corneal marker) remained stable (80.1±4.8%lacritin vs 85.6±1.8%; P>0.10). In addition, Aire(−/−) dry eye miceadministered LACRIPEP were also found to have less corneal staining,which is an indicator of cell death, as dry eye disease develops (FIG.4C; closed circles) relative to PBS (opened circles). Thus, topicallacritin, and its bioactive fragments thereof, diminished lacrimal glandinflammation and corneal staining in dry eye, and promoted ocularsurface differentiation. Importantly, suppression of inflammation andpromotion of tearing was achieved without direct contact withinflammatory cells nor with tear producing cells.

Topical lacritin stimulates tearing even without physical access tolacrimal acinar cells. The rapidity of the response is in keeping withcorneal sensory nerve activation. Individual corneal sensory nerveactivity was monitored at the level of the trigeminal ganglion in ratsvia previously described methods. Emerging from these studies was theobservation that topical lacritin is neural stimulatory. Topicallacritin enhanced the neural ‘dry response’, and to a lesser extent theneural ‘wet response’. The ‘dry response’ refers to neural activation asa consequence of drying of the cornea, which is thought to be a criticalTRPM8-mediated stimulus for tearing, while the ‘wet response’ occurswhen the agonist is present at the corneal nerve terminals. Neither ofthese responses was affected by negative control truncation mutant C-25,supporting the importance of the C-terminal α-helix in both neuralstimulation and tearing. It is likely that the enhanced dry response bylacritin, is due to a modulation of TRPM8 channels: ranging from a fullyinhibited TRPM8 state during wet cornea (with lacritin on board) byadrenergic α_(2A) and/or α_(2C) receptors to a completely disinhibited(activated) state during dry cornea (with lacritin removed). Apparentinhibition of the action potentials by lacritin during wet cornea wassmall because the TRPM8 activity during wet cornea is low to begin with,while it reaches optimal level during dry cornea when dynamic cooling ofthe ocular surface is taking place. Lacritin may also increase TRPM8neural density.

Stimulation of the dry response could be by indirect or directmechanisms. Lacritin stimulation of the corneal epithelium couldindirectly target sensory neurons via junctional-like complexes betweenthe two cell types. However, these are thought to be rare. Arguingagainst a direct mechanism are epithelial tight junctions that wouldimpede lacritin access to nerve endings. However Ca²⁺ and some growthfactors can loosen tight junctions. PDGF permeabilizes tight junctionsbetween cultured kidney cells within minutes, as does VEGF ofendothelial tight junctions, whereas chronic permeabilization of surfacecells of the stratified corneal epithelium is observed in MMP9- orinflammatory cytokine-linked inflammation and in bacterial infectionfrom endotoxin challenge. Lacritin dependent Ca²⁺ mobilization may besufficient to promote rapid and acute permeabilization for neuralaccess. We expect that a two-step process is involved. First, lacritinor lacritin peptide targeting of superficial corneal epithelial cellspromotes subtle loosening of tight junctions, perhaps by transientlyincreased trafficking of occludin into early endosomes, or by lacritindependent calcium signaling of the corneal epithelium since calciumregulates tight junction permeability. We expect that the process isactivated within 1 min, as per lacritin-stimulated calcium signalingwithin 20 sec, and lacritin stimulated autophagy by 1 min. In thismanner, lacritin or peptide gains entry. Subsequent neural stimulationmay be sufficient to trigger reclosure of tight junctions, as per theimportance of neural stimulation in corneal wound healing.

Syndecan-1 is a cell surface heparan sulfate proteoglycan that mediateslacritin targeting of cells, but only after heparanase (Ma et al, '06)has exposed GAGAL nestled among heparan sulfate chains. Heparanase alsogenerates heparan sulfate stubs that appear to be required for lacritinbinding, suggesting a hybrid GAGAL/heparan sulfate-binding site. Toassess the role of this interaction, cells were cultured overnight in4-methylumbelliferyl-b-D-xylopyranoside (‘xyloside’) to competitivelysuppress heparan and chondroitin sulfate assembly. Xyloside completelyabrogated lacritin cytoprotective activity. Thus, these activities aredependent on a region in its C-terminus that includes the syndecan-1binding domain. Further lacritin activities appear to be entirelyembodied within the sequences KQFIENGSEFAQKLLKKFS (‘N-94/C-6’; SEQ IDNO: 5) (Wang et al., 2006) or KQFIENGSEFAQKLLKKFSLLKPWA (‘N-94’; SEQ IDNO: 7) (Zhang et al., 2013) that when generated synthetically is aspotent as lacritin.

Lacritin targeting of corneal sensory neurons. Adrenergic α_(2C)selective antagonist MK912 inhibits lacritin accelerated autophagy inHCE-T cells, as does the syndecan-1 inhibitor xyloside. Both alsoinhibit lacritin stimulated FOXO3 phosphorylation. Activity profiles ofcorneal neurons before, during and 1 hr during/after 10 μM lacritinreveal a small inhibition (from ˜12 to ˜8 spikes/s) follows immediatelyafter the application of lacritin presumably due to α₂ adrenergicreceptor activation which inhibits TRPM8 channels. Removal of thisinhibition after 1 hr of lacritin and washout causes an enhancedexcitation of dry (from ˜20 to ˜23 spikes/s) and wet response (from ˜12to ˜16 spikes/s).

Example 2

The monomeric form of tear lacritin is a multifunctional factorresponsible for alleviating ocular surface stress. It is also an agonistfor basal tearing. Monomeric lacritin targets a heparanase (HPSE)deglycanated form of cell surface syndecan-1 (SDC1). However,polymerized lacritin and the lacritin-C splice variant are both unableto target SDC1 and are therefore inactive. We investigated whethereither SDC1 or HPSE might displace monomeric lacritin in dry eye tears,and if SDC1 or HPSE may be inadequate.

Methods:

Tears were collected onto Schirmer strips from 146 individuals before,and 1 day, 1 week and 1 month after photorefractive keratectomy. Tearswere stored at −70° C., and later eluted with a tear equivalent volumeof PBS, and pooled by time and normal (≥15 mm) vs dry eye (≤5 mm) tears.Tears were separated by SDS-PAGE and blotted with anti-N-terminalspecific lacritin mab 1F5, anti-C-terminal specific lacritin ab ‘abC-term’, anti-lacritin-C splice variant mab 4G6, anti-SDC1 mab A-38B,and with anti-heparanase abs #733 and #1453. Secondary abs wereprecleared over a tear column, and ab C-term was precleared over C-59lacritin truncation mutant.

Results:

Ab C-term detected less lacritin monomer in dry eye vs normal tears, adeficiency apparently compensated in dry eye by enhanced lacritin-Csplice variant. The 1F5 mab epitope is shared by both forms, and thusthe presumed hybrid band appeared greater in dry eye. Normal tears wereenriched in latent (uncleaved) HPSE and deglycanated SDC1. One day afterPRK, lacritin-C was further increased in dry eye, and both SDC1 and HPSEless in normals. Return to pre-PRK conditions was apparent by 1 month.

Conclusions:

Aqueous deficient dry eye tears are associated with decreased lacritinmonomer, increased lacritin-C splice variant, and less deglycanated SDC1and latent HPSE. These conditions are appropriate for the exacerbationor initiation of dry eye.

Example 3 Stability of the 25 Amino Acid C-Terminal Fragment of Lacritin

A limitation of most synthetic peptide drugs is their proteasesensitivity. Only HIV protease retropepsin and cathepsin K appearcapable of cleaving LACRIPEP according to PROSPER (Protease specificityprediction server) analysis, with the former cutting in the middle andthe latter removing the last alanine. Retropepsin would inactivateLACRIPEP, while cathepsin K would have no effect. However neitherprotease is found in normal human tears. Nonetheless, tears are rich inother proteases. We therefore incubated LACRIPEP in normal human tearsat 37° C. for 2, 4, 6 and 16 hr. For immunoblotting, we first removedall endogenous lacritin by immunodepletion. Remarkably, LACRIPEP wasstable for at least 16 hr as indicated in FIG. 6A, representingimmunoblots of a protease sensitive positive control ‘SN pep’ from adifferent protein and LACRIPEP (‘N-94’) after incubation inlacritin-depleted human tears for 2-16 hr at 37° C. Mass spectrometricanalysis of the SN pep, Lacripep (‘N-94’), and Lacripep without sixC-terminal amino acids (‘N-94/C-6’) demonstrated the relative stabilityof the three peptides after incubation in lacritin depleted tears for 4hr at 37° C. (FIG. 6B). Surprisingly, the smaller C-terminal fragment oflacritin (N-94/C-6; SEQ ID NO: 5) was found to be not as stable as theLACRIPEP peptide (SEQ ID NO: 7). Although mass spec analysis suggeststhat Lacripep (‘N-94’), and Lacripep without six C-terminal amino acids(‘N-94/C-6’) have similar stability in tears, and Lacripep without sixC-terminal amino acids (‘N-94/C-6’) was stable in phosphate bufferedsaline for 29 days at 62° C. (FIG. 6B), immunoblotting reveals thatN-94/C-6 loses epitopes after incubation in lacritin depleted tears for4 hr at 37° C. whereas Lacripep (‘N-94’) does not. Accordingly, thefinal 6 amino acids of native lacritin have relevance in enhancing thestability of bioactive fragments of lacritin making N-94 a superiorpharmaceutical peptide relative to N-94/C-6.

Another advantage of LACRIPEP is its low dose optimum. In human cellculture its optimal dose is 1-10 nM. In animal studies, ˜4 μM (0.0012%)is optimum (FIGS. 7A & 7B). 4 μM LACRIPEP has also been found to bebactericidal, but not hemolytic. Lacritin as a whole protein does nothave bactericidal activity.

To monitor LACRIPEP in whole body toxicity studies, LACRIPEP wassynthesized with a single C-terminal tyrosine for iodination to form¹²⁵I-Lacripep-Y. Rats administered a single 4 μM dose of ¹²⁵I-Lacripep-Ydemonstrated high retention in eye tears with minimal levels detected inthe blood and serum (see FIG. 8).

Example 4 A Cleavage-Potentiated Fragment of Tear Lacritin isBactericidal

Experimental Procedures

Tears and Tear Immunodepletion—Normal human basal tears were collected.Briefly, tears from 0.5% proparacaine anesthetized eyes were collectedonto preweighed wicks and flash-frozen for “70° C. storage. Tears wereeluted by immersion of each strip in 30 #1 of PBS for 20 min, followedby centrifugation. For immunodepletion, 10-fold diluted tears wereincubated overnight (4° C.) or for 1 h at room temperature with proteinA beads (0.2 ml, NAb Spin Kit, Peirce/Thermo Scientific) saturated with“anti-N-65 Lac C-term” or preimmune Ig. N-65 is a lacritin truncationmutant lacking 65 N-terminal amino acids. The tear flow-through aftercentrifugation (5000 # g for 1 min) was then assayed for antibacterialactivity.

Lacritin Constructs, Purification, Synthetic Peptides, and MassSpectrometry—Lacritin N-terminal truncations N-55, N-65, N-71, and N-75were generated by PCR from parent cDNA pLAC, as described previously(Zhang et al, (2013) J. Biol. Chem. 288, 12090-12101). N-terminaldeletions of 80 (N-80) and 86 (N-86) amino acids were generated usingforward primers GGTGGTCATATGAAAGCAGGAAAAGGAATGCACGG (SEQ ID NO: 9) andGGTGGTCATATGCACGGAGGCGTGCCAGGTGG (SEQ ID NO: 10) 3, respectively, andcommon reverse primer GGTGGTCATATGTATATCTCCTTCTTAAAG (SEQ ID NO: 11).All constructs were verified by sequencing. Bacterial protein expressionand purification of recombinant lacritin and lacritin truncations wereperformed as described previously (Zhang et al, (2013) J. Biol. Chem.288, 12090-12101). Briefly, cleared cell (ER2566 or BL21-CP) lysateswere loaded on chitin columns (IMPACT-CN System; New England BiolabsInc., Beverly, Mass.) equilibrated with 50 mM Tris, 0.5 M NaCl (pH 8),followed by 20 column volumes of washing, elution with 50 mM2-mercaptoethanol for 16 h at room temperature in the same buffer,extensive dialysis against PBS (4° C.), and protein quantitation.Further DEAE purification removed a ˜9-kDa lacritin proteolytic fragmentand bacterial contaminants in which lacritin was collected as the flowthrough with 140 mM NaCl in phosphate buffer, pH 7.2. Synthetic peptidesN-80/C-25, N-94, N-94/C-6, N-94/C-10, N-94/C-15, N-99, and N-104 weresynthesized by Genscript (Piscataway, N.J.) with acetylated Ntermini.Purity was 95%. C-termini of all were amidated, with the exception oflacritin C-terminal N-94, N-99, and N-104. N-64/C-31 was neitheramidated nor acetylated and was synthesized by the University ofVirginia Biomolecular Research Facility. The nature of the lacritin˜9-kDa fragment was pursued by Western blotting. Briefly, lacritinbefore and after DEAE separation was separated by SDS-PAGE and thentransferred and blotted with anti-Pep Lac N-terminal and anti-N-65 LacC-terminal antibodies, respectively diluted 1:200 or 1:400 in PBScontaining 0.3% Tween 20. Detection was with ECL.

For fragment purification, chitin-enriched lacritin was dialyzed againstphosphate buffer containing 14 mM NaCl (pH 7.2). Following incubationwith DEAE equilibrated in the same buffer, the ˜9-kDa fragment wascollected in the flow-through, whereas intact (18 kDa) lacritin waseluted with 140 mM NaCl in phosphate buffer, pH 7.2. After determinationof protein concentration (BCA assay), both were aliquoted, lyophilized,and stored at −70° C. Analysis was by SDS-PAGE on 4-20% gradient gels.The identity of the ˜9-kDa fragment was determined by mass spectrometry.

Bacterial Growth, SYTOX Green Assays, and on Column Cleavage—E. coli(ATCC (Manassas Va.) catalog no. 10536), S. epidermidis (ATCC catalogno. 12228), and P. aeruginosa (ATCC catalog no. 9027) were grown tomid-log phase in 50 ml of Luria-Bertani (LB) medium and washed threetimes in phosphate buffer containing 10 mM NaCl (pH 7.2; PB—NaCl) withcentrifugation. Pellets were resuspended in 1 ml of PB—NaCl.

For lacritin inhibition assays, 50 ul of bacterial pellets each diluted1:100 in PB—NaCl were incubated for 1.5 h (37° C.) with 100 ul oflacritin, lacritin truncations, or synthetic peptides at a finalconcentration of 0.1-6 uM. Mixtures were diluted 1:10 in PB—NaCl beforeplating 100 ul in quadruplicate on LB agar plates for overnight growthat 37° C. Colonies were manually counted. In other experiments, mid-logE. coli was treated at 37° C. for 0, 1, 2, or 3 h with 2 uM lacritin orlacritin truncations or with ampicillin (5 uM) or tetracycline (2 uM).After each treatment, 100 ul was centrifuged, resuspended in 1 ml ofPB—NaCl, and plated (100 ul) onto LB agar for overnight growth (37° C.)and colony counting.

For salt sensitivity studies, pelleted and washed mid-log phase E. coli,S. epidermidis, or P. aeruginosa were resuspended in 1 ml of PB—NaCl andthen treated as above with PB—NaCl or with 3 uM N-65 in 130, 280, or 380mosmol/liter PB—NaCl for 1.5 h (37° C.). Mixtures were diluted 1:10 inPB—NaCl before plating 100 ul of each in quadruplicate on LB agar platesfor overnight growth at 37° C. Colonies were manually counted.

For bacterial permeability assays, pelleted and washed midlog phase E.coli were resuspended in 1 ml of PB—NaCl and then treated as above with3 uM lacritin, N-65, or C-25 or with 10% Triton X-100. Similarly, washedmid-log phase S. epidermidis were resuspended in 1 ml of PB—NaCl andthen treated with lacritin or C-25 or a ˜9-kDa purified lacritinfragment. Later, 1 ul of 0.5 mM SYTOX Green was added to each well of96-well fluorescent microtiter plates. Readings were taken at 5-minintervals at respective excitation and emission wavelengths of 485 and538 nm using a Fluoroskan Ascent FL fluorometer (Thermo FisherScientific). In parallel, SYTOX Green internalization was visualized byconfocal microscopy after 1 h of 10% Triton X-100, PB—NaCl, or 3 uM N-65treatment of washed mid-log phase E. coli.

For cell-free synthesis without glycosylation, full-length lacritin cDNAin pLacSL was PCR-amplified and subcloned into pTXB1 supplied by themanufacturer (New England Biolabs, Ipswich, Mass.). Cell-free synthesisand subsequent removal of ribosomes, followed by metal affinity resinadsorption of His-tagged factors, was performed as per themanufacturer's instructions (New England Biolabs; PURExpress)Immediately following expression, an aliquot was stored at −60° C. Otheraliquots were incubated at 37° C. for 24 and 48 h. Each was separated bySDS-PAGE, transferred, and blotted with anti-N-65 Lac C-terminalantibodies.

For lacritin cleavage assays, supernatants from saturated 50-mlovernight cultures of S. epidermidis were collected by centrifugation(10 min; 11,000 rpm). Each supernatant was then incubated for 4, 16, and20 h (37° C.) in PB—NaCl with chitin beads containing lacritin-inteinimmobilized via N-termini C-terminal cleavage products were collected byPBNaCl washing, separated by SDS-PAGE, transferred, and blotted withanti-N-65 Lac C-terminal antibodies. In some experiments, supernatantsand lysates from overnight cultures of S. epidermidis, Staphylococcusaureus, P. aeruginosa, and E. coli were incubated overnight (37° C.)with lacritin in solution in PB—NaCl. Mixtures were then separated bySDS-PAGE, transferred, and blotted with anti-N-65 Lac C-terminalantibodies. Parallel studies monitored the integrity ofchitin-intein-immobilized lacritin in PB—NaCl at 37° C. for 0, 24, 48,and 72 h or for 24 h (37° C.) with 1 uM pepstatin, 10 uM bestatin, 100uM antipain, 1 mM 4-benzenesulfonyl fluoride hydrochloride, 100 uMchymostatin, 10 uM E64, 100 uM leupeptin, or 10 mM phosphoramidon or for24 h after boiling for 5 min at 100° C.

Hemolysis Assay—The method of Cerovsky' et al. was followed with somemodifications. Washed sheep red blood cell pellets (MP Biomedicals,Santa Ana, Calif.) were suspended for 1 h at 37° C. in 565 ul of PBSplus 100 ul of lacritin, N-55, N-65, N-71, N-75, N-80, or C-25 at afinal concentration of 2 uM or with N-65, N-64/C-31, N-80/C-25, N-94,N-94/C-6, N-94/C-10, N-94/C-15, N-99, or N-104 at a final concentrationof 6 uM. As respective positive and negative controls, Triton X-100(final concentration of 5%) or PBS was included in place of lacritin orlacritin fragments. After centrifugation (250×g; 5 min), the absorbancesof supernatants at 540 nm were monitored.

Metabolome Analysis—Washed mid-log E. coli were incubated with 6 uM N-65or PB—NaCl for 15 min at 37° C. in replicates of six, each at 1×10⁸cells/replicate. Cells were then washed once, and pellets wereflash-frozen for storage at −70° C. Unbiased metabolite analysis wasperformed by Metabolon Inc. (Durham, N.C.) using GC/MS and LC/MS/MS. 78metabolites were identified.

Statistical Analyses—With the exception of the single metabolomicanalysis, all experiments were performed at least three times.Statistical analysis of metabolite data was performed, where raw datavalues were first log transformed to be closely distributed as a normaldistribution and then assessed by a non-parametric Wilcoxon test andtwo-sample t test. For both tests with p & 0.05, metabolites wereconsidered significantly different and further analyzed by hierarchicalclustering for their association patterns. Data are reported as themean+/−S.E.

Results

Lacritin Bactericidal Activity in Tears—Tears protect the surface of theeye against environmental pathogens and are enriched in the prosecretorymitogen lacritin, which flows onto the eye during basal and reflextearing. Lacritin is 21% identical to dermcidin, whose proteolyticallyprocessed C terminus contributes to the bactericidal activity of humansweat. We sought to determine whether lacritin or a lacritin fragment(s)have bactericidal activity. Half-diluted basal tears completely blockedE. coli growth and E. coli is a significant contributor to bacterialconjunctivitis in the developing world, as is P. aeruginosa. We testedtears that had been passed over immobilized anti-N-65 Lac C-terminalantibodies (ab C-term) to immunodeplete both lacritin and C-terminallacritin fragments, or over preimmune Ig (mock-depleted). Both werediluted 10-fold for dose-dependent challenge of E. coli and P.aeruginosa. Mock-depleted tears suppressed E. coli and P. aeruginosacolonies in a tear volume-dependent manner. This contrasted withC-terminal antibody-immunodepleted tears, which were as ineffective asthe phosphate buffer negative control.

Lacritin's C Terminus Contains a Bactericidal Domain—Lacritin's Cterminus contains three predicted α-helices each validated by circulardichroism. The most C-terminal α-helix is amphipathic and targetssyndecan-1 as an initiator of corneal epithelial cell proliferation andsurvival, largely via hydrophobic face residues. Association ofamphipathic α-helices with bacterial membranes can be destabilizing. Toexplore whether these or other lacritin domains are bactericidal, wegenerated recombinant lacritin and lacritin truncations. Each wasgenerated as an intein fusion protein, purified on chitin to also removethe intein tag and then on DEAE to exclude bacterial contaminants.Lacritin and truncations were then assayed in equimolar (2 uM) amountsin the presence of mid-log E. coli, P. aeruginosa, or S. epidermidis. P.aeruginosa is an eye pathogen often responsible for keratitis in contactlens wear. S. epidermidis is a common cause of conjunctivitis andkeratitis and is abundant in blepharitis, an eyelid inflammationassociated with slightly altered tear composition, including selectivelyless lacritin. Lacritin without truncation had no effect on theappearance of colonies, with numbers equivalent to the phosphate buffernegative control. However, few colonies were apparent with lacritinlacking 65 (N-65) or 80 (N-80) amino acids from the N-terminus, aneffect completely or partly negated by removing six additional aminoacids (N-86) in E. coli or P. aeruginosa but not S. epidermidis. Aminoacids 81-86 comprise the sequence LAKAGKG (SEQ ID NO: 12), which alignswith a sequence in a potent dermcidin fragment SSL-25 with an amino acididentity of 44%.

To determine whether the LAKAGKG (SEQ ID NO: 12) region was responsible,we generated AKAGKGMHGGVPGG (SEQ ID NO: 13; amino acids 81-94;N-80/C-25), comprising the truncation-narrowed portion of the SSL-25homologous region. Also generated were partially overlappingLKSIVEKSILLTEQALAKAGKGMH (SEQ ID NO: 14; amino acids 65-88; N-64/C-31)and C-terminal KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7; amino acids95-119; N-94). Unexpectedly, colonies were abundant with N-80/C-25 andN-64/C-31, whereas few or no colonies were apparent with N-94, a regiononly 12.5% identical with the C-terminus of dermcidin. To narrow thissite, we generated synthetic peptides with amino acids sequentiallyremoved from the carboxy terminus N-94/C-6, N-94/C-10, N-94/C-15 andN-99 ENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 15), and N-104 (FAQKLLKKFSLLKPWA(SEQ ID NO: 16). N-94 and N-104 were fully active but not the otherpeptides, although N-94/C-6 (SEQ ID NO: 5) was slightly so. N-65 isbactericidal and equipotent to ampicillin. In dose response studies,N-104 was almost as effective as N-65, with a half-maximal inhibition ofabout luM for E. coli and about 1-1.5 uM for P. aeruginosa, a dose rangecommon to antimicrobial peptides.

DISCUSSION

The rationale for exploring whether lacritin might be bactericidal wasits 21% identity with dermcidin, whose proteolytically processed Cterminus contributes to the bactericidal activity of human sweat and isin tears. Surprisingly, dermcidin primary sequence homology was not thesource of lacritin activity. Only 40.7% identity is shared betweendermcidin's bactericidal SSL-25 peptide and the homologous lacritinregion that as a synthetic peptide was inactive. Instead, lacritin N-104fragment with 7% dermcidin identity embodies the core activity, a hybriddomain consisting of an N-terminal amphipathic helix and hydrophobicC-terminal coiled coil tail, together appropriate for bacterial membranecontact and insertion, as was apparent by rapid entry ofmembrane-impermeable SYTOX Green in N-65-treated cells. Surprisingly, aC-terminal 25 amino acid fragment KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO:7; amino acids 95-119; N-94) was found to be fully active, whereinremoval of 6 terminal amino acids (e.g. KQFIENGSEFAQKLLKKFS; SEQ ID NO:5) substantially reduced the bactericidal activity of the peptide.

Example 5

Although topical application of ophthalmic products has remained themost popular and well-tolerated administration route for patientcompliance, the bioavailability of eye drops is severely hindered byblinking, baseline and reflex lacrimation, and nasolacrimal drainage.One solution to enhance the therapeutic index of topical treatments isthrough the application of polymeric nanoparticles as drug carriers.

One solution to enhance the therapeutic index of topical treatments isthrough the application of polymeric nanoparticles as drug carriers.Polymeric nanoparticles displaying therapeutic ligands at the corona caninteract with complex biomolecular architectures through multiplesimultaneous interactions (multivalency) and exhibit the well-definedsizes required for efficient tissue penetration. One such materialcapable of being employed as the scaffold are thermo-responsiveelastin-like polypeptides (ELPs). ELPs are composed of the repetitivepentapeptide motif (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 24) and exhibitunique reversible inverse phase transition temperatures, Tt, below whichthey solubilize and above which they phase separate. Tt can be modulatedthrough guest residue (Xaa) selection and changes in the number ofpentameric repeats, n.

Inspired by the motivation to further explore lacritin's function on theocular surface, enhance its bioavailability, and better target thecorneal epithelium, we utilized a diblock ELP (SI) nanoparticle scaffoldto bioengineer LSI nanoparticles with multivalent presentation oflacritin at the surface.

Materials and Methods

Materials and Equipment

TB DRY® Powder Growth Media was purchased from MO BIO Laboratories, Inc.(Carlsbad, Calif.). NHS-rhodamine was purchased from Thermo FisherScientific (Rockford, Ill.). SV40-Adeno vector transformed cornea cells(RCB 2280, HCE-T) were purchased from Riken Cell Bank, Japan.Keratinocyte-SFM medium supplied with Bovine Pituitary Extract (BPE) andprequalified human recombinant Epidermal Growth Factor 1-53 (EGF) waspurchased from Gibco Invitrogen (Life Technologies, NY). CalciumIndicator Fluo-4, AM, cell permeant was purchased from Life Technologies(NY). Algerbrush II with a 0.5 mm burr was purchased from The AlgerCompany, Inc., TX. In vivo studies were conducted using in house bred 12week female non-obese diabetic (NOD) (Taconic Farms, Germantown/NY, USA)mice.

Construction of LSI Nanoparticles

Genes encoding for ELPs (SI) were synthesized by recursive directionalligation in pET25b(+) vector. A sequence encoding human lacritin withoutsecretion signal peptide was designed using the best E. coli codons inEditSeq (DNAStar Lasergene, WI). A thrombin cleavage site was designedbetween the lacritin sequence and ELP tag via insertion at the B seRIsite. Lacritin gene flanked by NdeI and BamHI restriction digestionsites at the 5′ and 3′ ends was purchased in the pIDTSmart-KAN vectorfrom Integrated DNA Technologies (IDT) as follows:

(SEQ ID NO: 25) CATATGGAAGACGCTTCTTCTGACTCTACCGGTGCTGACCCGGCTCAGGAAGCTGGTACCTCTAAACCGAACGAAGAAATCTCTGGTCCGGCTGAACCGGCTTCTCCGCCGGAAACCACCACCACCGCTCAGGAAACCTCTGCTGCTGCTGTTCAGGGTACCGCTAAAGTTACCTCTTCTCGTCAGGAACTGAACCCGCTGAAATCTATCGTTGAAAAATCTATCCTGCTGACCGAACAGGCTCTGGCTAAAGCTGGTAAAGGTATGCACGGTGGTGTTCCGGGTGGTAAACAGTTCATCGAAAACGGTTCTGAATTCGCTCAGAAACTGCTGAAAAAATTCTCTCTGCTGAAACCGTGGGCTGGTCTGGTTCCGCGTGGTTCTG GTTACTGATCTCCTCGGATCC.The above gene was subcloned into the pET25b(+) vector and the LSI genewas synthesized by ligation of ELP SI gene via the BseRI restrictionsite. Correct cloning of the fusion protein gene was confirmed by DNAsequencing. LSI fusion proteins were expressed in BLR (DE3) E. coli(Novagen Inc., Milwaukee, Wis.) for 24 h in an orbital shaker at 37° C.at 250 rpm and purified via inverse phase transition cycling.

Characterization of LSI Phase Behavior and Nanoparticle Formation

The phase diagram for LSI fusion protein was characterized by opticaldensity change at 350 nm as a function of solution temperature using aDU800 UV-Vis Spectrophotometer (Beckman Coulter, Brea, Calif.). Tt wasdefined at the point of the maximum first derivative. Self-assembly ofnanoparticles was measured using dynamic light scattering (DLS) using aDynaPro-LSR Plate Reader (Wyatt Technology, Santa Barbara, Calif.).Light scattering data were collected at regular temperature intervals(1° C.) as solutions were heated from 5 to 50° C. The results wereanalyzed using a Rayleigh sphere model and fitted into a cumulantalgorithm based on the sum-of-squares value. The critical micelletemperature (CMT) was defined as the lowest temperature at which the Rhis significantly greater than the average monomer Rh.

TEM Imaging of LSI Nanoparticles

The TEM imaging was carried out on a FEI Tecnai 12 TWIN microscope(Hillsboro, Oreg.) at 100 kV. Briefly, a 100 uM solution (5 uL) wasinitially deposited on a copper grid with carbon film (CF400-Cu,Election Microscopy Sciences, Hatfield, Pa.). After removing the excessamount of solution with filter paper, the samples were negativelystained with 2% uranyl acetate, followed by removing excess uranylacetate after 30 s. The samples were then dried under room temperaturefor at least 3 h before use in imaging.

SV40-Immortalized Human Corneal Epithelial Cell (HCET) Culture

SV40-immortalized HCE-T cells (Riken Cell Bank, Japan) were grown inkeratinocyte-SFM media (KSFM, Life Technologies, Rockville, Md.)containing bovine pituitary extract (BPE, 50 mg/ml) and epidermal growthfactor (EGF, 5 ng/ml). Cell passages 4-6 were used for Ca2+ imaging,scratch and uptake assays in 35 mm coverslip-bottomed dishes. Tooptimize responsiveness upon stimuli, cells were starved with EGF andBPE free medium for 24 h before experimentation.

Ca2+ Imaging

HCE-Ts were rinsed twice with Ca2+ and Mg2+ free phosphate buffer saline(PBS) and incubated at 37° C. for 20 min fresh KSFM medium containing2.5 mM calcium probe Fluo-4AM (Invitrogen Life technologies, NY). Thecells were then rinsed twice with NaCl Ringer buffer (145 mM NaCl, 5 mMKCl, 1 mM CaCl₂, 1 mM KH2PO4, 1 mM MgCl₂, 10 mM glucose, and 10 mMHEPES, osmolarity 300, pH 7.4) and kept in the same buffer at roomtemperature for 30 m. For Ca2+ free medium, 1 mM Ca2+ was replaced with0.5 mM EGTA. The cells were illuminated at 488 nm, and their emissionwas monitored every 3.15 s at 510 nm using Zeiss LSM 510 Meta confocalmicroscope system. The field of interest contained 24 to 45 cells, andthe fluorescent intensity change was calculated for each region withimage-analysis software. Ca2+ dynamics were evaluated using the changesin fluorescence intensity of Fluo-4AM. The data are presented aspercentage change in fluorescence intensity at each time point (Ft) tothe first time point (F0) reading: (Ft−F0)/F0×100%.

In Vitro Scratch Closure Assay

For a scratch assay, confluent HCE-T monolayers were scraped in astraight line to create a scratch wound with a p200 pipet tip. Cellswere rinsed with KSFM medium without BPE or EGF to remove debris andthen incubated with fresh KSFM medium containing BPE (50 mg/mi) and EGF(5 ng/ml), LSI, or medium without growth factors (No treat). Phasecontrast images of the wound at the beginning and after 24 h treatmentwere captured using Zeiss LSM 510 Meta confocal microscope system.

Exogenous Cell Uptake Assay

SI and LSI nanoparticles were conjugated with NHS-rhodamine (ThermoFisher Scientific Inc, Rockford, Ill.) via covalent modification of theamino terminus. Conjugation was performed in 100 mM borate buffer (pH8.0) for 2 h (LSI) or overnight (SI) at 4° C. followed by desalting on aPD10 column (GE Healthcare, Piscataway, N.J.) to remove free dye.Briefly, after the cells were rinsed with fresh medium without BPE andEGF, 10 mM rhodamine labeled proteins were added into the dish. Afterincubation at 37° C. for different time points, the cells were rinsedand images were acquired using Zeiss LSM 510 Meta confocal microscopesystem.

Murine Corneal Abrasion and Recovery Study

Briefly, 12 week female NOD mice were anesthetized with an i.p.injection of xylaxine/ketamine (60-70 mg+5 mg/kg) and placed on aheating pad. After cleaning the ocular surface with eye wash (OCuSOFT,Inc., TX), the corneal epithelium of the right eye was removed down tothe basement membrane using an algerbrush II (The Alger Company, Inc.,TX); the left eye was left intact as a contra lateral control. Mice wereallowed to heal for 24 h with 2 doses (5 ml) of KSFM medium containingBPE (50 mg/ml) and EGF (5 ng/ml), 100 mM LSI, 100 mM SI, or no treatmentat 12 h intervals. After staining the ocular surface with 5 ml 0.6 mg/mlfluorescein (Akorn, Ill.), images of the abrasion wound were capturedusing a Moticam 2300 camera after 12 h and 24 h.

Statistics

All experiments were replicated at least three times. Maximumfluorescence intensity change in Ca2+-mediated fluorescence was analyzedusing a non-paired t-test. Scratch wound healing quantification wasanalyzed using a one-way ANOVA followed by Tukey's post hoc test. HCE-Tuptake was analyzed using two-way ANOVA followed by Bonferroni post-testand murine corneal epithelium recovery from abrasion wound were analyzedusing Kruskal-Wallis non-parametric ANOVA. Corneal wound healingcomparison between LSI and LS96 after 12 h treatment was analyzed usingMann-Whitney U test. A p value less than 0.05 was consideredstatistically significant.

Results and Discussion

The ELP lacritin fusion called LSI forms thermoresponsive nanoparticles

Two derivatives of lacritin were formed, each comprising an ELP tag:

LSI (SEQ ID NO: 26) GEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWAGLVPRGSG(VPGSG)₄₈(VPGIG)₄₈Y; and LS96 (SEQ ID NO: 27)GEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWAGLVPRGSG(VPGSG)₉₆Y.

LSI and LS96 were cloned into a pET25(+) vector, expressed in E. coli,and purified using inverse phase transition cycling. LSI was expected toundergo thermally-mediated assembly similar to SI and form nanoparticlesabove its phase transition temperature (Tt), while LS96, with lacritingene fused to the soluble macromolecule S96, was developed as a controlthat does not phase separate until significantly above physiologicaltemperatures. After confirming the purity and molecular weight ofexpressed proteins, their phase diagrams were characterized usingoptical density as a function of temperature. While monomeric ELPsundergo a single phase transition from solubility to coacervate, certainELP diblock copolymers display two steps of assembly in response toheating: (i) soluble monomers assemble into stable nanoparticles aboveTt1; and (ii) at a higher temperature, Tt2, the nanoparticles themselvescoacervate. For ELPs such as LSI, Tt1 is thus defined as the criticalmicelle temperature (CMT) above which nanoparticles are favorable (32.3°C. at 25 mM). Tt2, or the bulk phase transition temperature, representsthe temperature at which these nanoparticles further assemble intocoacervates. In striking contrast to its SI scaffold, LSI only shows onephase transition at 18.4° C. (25 mM). Moreover, LSI illustrated lessconcentration dependent phase transition compared to the SI scaffold, asdemonstrated by a decreased slope when Tt was fit by the equation: Tt=mlog[C_(ELP)]+b, where C_(ELP) is the concentration, m is the slope, andb is the transition temperature at 1 mM. Eqn (1) permits the estimationof Tt over a broad range of concentrations, which may be encountered invivo. In our recent reports, suppression of the ELP concentrationdependence correlates with assembly mediated by the fusion domainitself, which we have reported in fusion between a single chain antibodyand also a disintegrin. Based on the unexpected observation that LSIexhibits a single phase transition, dynamic light scattering (DLS) wasused to determine whether particles form above or below this Tt.

Both constructs were thus compared by DLS to monitor the temperaturedependent assembly process. Surprisingly, LSI preassembled into 30-40 nmnanoparticles even below Tt. Above Tt, it began to favor largernanoparticles ranging from 130-140 nm. SI remained as 20-30 nm micellesat physiologically relevant temperatures. In combination with theoptical density data, this suggests that lacritin itself mediatespartial assembly of small aggregates that proceed to assemble largerstructures above the Tt1 mediated by SI. To further examine the dominantstructures formed by LSI and SI, we observed their morphologies whendried from room temperature using transmission electron microscopy(TEM). Consistent with DLS, while SI formed a mono-dispersed micellestructure with an average diameter of 36.5+/−5.8 nm and LSI formedlarger nanoparticles that exhibit average diameters of 67.1+/−11.5 nm.Regardless, both SI and LSI appear capable of forming nanostructures.

LSI Nanoparticles Exhibit Mitogenic Activity Using SV-40 TransducedHuman Corneal Epithelial Cells.

Upon injury, one of the earliest reactions of many epithelial cells is atransient Ca2+ wave spreading across the monolayer cell sheet. The Ca2+wave triggers downstream signaling pathways responsible for cellmigration, proliferation and other events associated with wound repair.Lacritin has been reported as stimulating Ca2+ wave propagationthroughout HCE-Ts and further studies have confirmed that this Ca2+signal is associated with lacritin's protection of HCE cells stressedwith benzalkonium chloride and maintenance of cultured corneal epitheliahomeostasis. To confirm whether LSI maintains mitogenic activity oflacritin, we tested both calcium transients and scratch wound healingassays based on the reported HCE-T model. We first tested intracellularCa2+ wave propagation in HCE-T cells loaded with Fluo-4 AM under eitherLSI or SI treatment. The fields of interest containing 24 to 45 cellswere chosen and the fluorescent intensity change of ten individual cellswas calculated using LSM 510 image-analysis software. Percentage changein fluorescence intensity at each time point (Ft) to the first timepoint (F0) reading: (Ft−F0)/F0×100% was used to quantify Ca2+ signal.The signal triggered by SI was negligible, evoking only a 0.054+/−0.049fold maximum fluorescence intensity change compared to baseline. Theaddition of LSI nanoparticles, however, resulted in a significantlyrapid calcium influx into the cells with a maximum fluorescenceintensity 4.399+/−1.043 fold of F0 (p<0.0001). Moreover, HCE-T cellsappeared to have ‘memory’ for exogenous LSI treatment, as treating thesame group of cells for the second time with the same concentrationresulted in a broader peak for Ca2+ influx, which extended peak durationfrom 40 to 70 s. Downstream of Ca2+ mediated signaling, HCE-Ts are knownto initiate more rapid motility and proliferation, which can bevisualized during the closure of a scratch made on a confluent sheet ofcells.

To visualize the in vitro effect of LSI, we applied a scratch to a sheetof cells and captured the timelapse healing process. Each treatment wasperformed in triplicate and four independent wound distances in eachwell were measured for analysis. After 24 h of treatment, a very lowconcentration of LSI (10 nM) significantly accelerated scratch woundhealing compared to plain medium without growth factors (***p<0.001).This effect was comparable to a positive control containing BPE and EGF.

LSI Nanoparticles Undergo Uptake into HCE-Ts

Encouraged by LSI's in vitro mitogenic activity, we further exploredwhether exogenous LSI can enter the HCE-Ts. The cells were thusincubated with NHS-rhodamine labeled LSI and SI nanoparticles fordifferent time points. Consistent with lacritin-mediated uptake, LSIunderwent cell uptake into HCE-Ts in a time dependent manner Significantcell entry was observed 10 m following incubation, and after 1 h, LSInanoparticles accumulated within the peri-nuclear region. Uponquantification, LSI exhibited significantly higher cytosolicfluorescence than SI nanoparticles (p<0.0001). Nanomaterials ofdifferent sizes, shapes, and charges have been widely used in biomedicalimaging, tissue targeting, and cell uptake. More recently, the use ofnanoparticles to crosslink membrane receptors more efficiently toregulate downstream signaling has attracted enormous attention,especially in antibody mediated receptor crosslinking.

LSI Nanoparticles Heal a Corneal Abrasion on Non-Obese Diabetic (NOD)Mice

We proceeded to investigate LSI nanoparticles in vivo efficacy viatopical eye drops. In this study, we developed a corneal epithelialabrasion model on female NOD mice to assess the wound-healing effect ofLSI nanoparticles. Non-obese diabetic (NOD) mice are frequently used asan animal model for impaired wound healing in humans. Reduced cellproliferation, retarded onset of the myofibroblast phenotype, reducedprocollagen I mRNA expression, and aberrant control of apoptotic celldeath were observed in NOD group. The NOD mouse model was selected forevaluation of the in vivo activity of LSI nanoparticles. Brifly, acircular abrasion wound with a diameter of around 2 mm was created onthe right eye of the animal with an algerbrush II without damaging thelimbal region Immediately after imaging, 5 ml of 100 mMLSInanoparticles, SI nanoparticles, or control EGF+BPE were topicallyadministered to the ocular surface, and this treatment was repeated once12 h after wound initiation. Images of the wound were captured at time0, 12 h, and 24 h using fluorescein staining under cobalt blue light.The initial wound healing comparison study included 4 mice under eachtreatment group, with the left eye intact as a contralateral control.After experimentation, wound-healing images were analyzed using ImageJ.Mean fluorescein intensity, wound area, total fluorescein (total=meanfluorescein intensity×wound area), fluorescein percentage of initialvalue, wound area percentage of initial value (PctArea), and totalfluorescein percentage of initial value were determined by a blindreviewer and compared between groups at 12 h and 24 h usingKruskal-Wallis non-parametric testing. No significant inflammation orany other adverse effects were observed upon treatments. Notably, LSI atboth 12 and 24 hours significantly decreased the percentage of initialwound area (PctArea) compared to SI (p=0.001), EGF+BPE (p=0.001), and notreatment groups (p=0.001), suggesting that LSI is the best formulationto accelerate recovery of the corneal epithelium. To corroborate thefluorescein imaging result, we further processed the corneal epitheliumafter 24 h for histology analysis. Briefly, corneas were fixed,sectioned across the defect, and stained by hematoxylin and eosin.

Pathology of Corneal Epithelium (EP);

Bowman's membrane (BM); stroma (ST); Descenet's membrane (DM);endothelium (EN) was evaluated. Remarkably, the corneal epithelium ofthe LSI treatment group showed complete recovery with a smooth,reconstituted surface, absent of inflammation. While the fluoresceintest revealed partial resistance to staining at 24 h in the SI group,the regenerated corneal epithelium did not complete differentiation.Having demonstrated that the mitogenic lacritin protein remains activewhen decorated on a protein polymer nanoparticle, we next investigatedwhether ELP-mediated particle assembly is required to achieve thisresult. To address the significance of ELP assembly in vivo, theefficacy of LSI nanoparticles can be directly compared with a thermallynonresponsive lacritin fusion protein called LS96. Both LSI and LS96contain the lacritin sequence followed by an ELP containing 96 totalpentameric repeats; however, the ELP S96 does not phase separate untilabove physiological temperatures. Optical density measurements, in fact,revealed that LS96 does not display any observable phase transitions inphosphate buffered saline. In addition, DLS confirmed that LSI has amuch larger hydrodynamic radius than LS96 at 37° C.

Using these two related formulations of lacritin ELPs, the cornealdefect study in NOD mice was both to confirm the ability of LSI to closethe epithelium after 12 h and compare this closure with that of LS96. Tobetter evaluate our experimental observation, we further increased thesample size to eight mice per group, with all right eyes receiving theabrasion procedure. Interestingly, LSI healed the abrasion woundsignificantly (p<0.05) faster than the non thermo-responsive LS96fusion. This finding directly supports the contention that ELP-mediatedassembly is involved with the enhancement of LSI.

CONCLUSIONS

To accelerate the corneal wound healing process, a multivalent ELPnanoparticle was used as a means of delivering a candidatebiopharmaceutical, the mitogen lacritin, to the ocular surface. Thislacritin ELP fusion, LSI, displays thermoresponsive self-assemblyproperties similar to the unmodified SI nanoparticle and presentsaccessible lacritin at its corona at physiologically relevanttemperatures. LSI nanoparticles trigger calcium dependent cellsignaling, internalize into cells, and facilitate scratch closure inmonolayers of a human corneal epithelial cell line (HCE-Ts). Whentopically applied on the ocular surface of NOD mice following removal ofthe corneal epithelium, LSI nanoparticles promoted faster wound healingcompared to SI and untreated groups. Most importantly, the LSInanoparticles produce faster regeneration of the corneal epitheliumcompared to a control lacritin ELP fusion, called LS96, that does notundergo thermally-mediated assembly. Overall, this study highlights thepotential of ELPs as nanoparticle scaffolds to effectively deliverprotein therapeutics to the ocular surface and repair abrasion wounds.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

1-20. (canceled)
 21. A bactericidal composition, comprising a C-terminalfragment of lacritin selected from SEQ ID NO: 7, or SEQ ID NO: 8, or aderivative thereof that differs from SEQ ID NO: 7 or SEQ ID NO: 8 by oneor two amino acid substitutions; and a pharmaceutically acceptablecarrier; wherein the composition is suitable for topical administrationto an ocular surface of a subject.
 22. The composition of claim 21wherein the amino acid substitutions are located at positions 4, 6, 8,10, 17 and 19 relative to the numbering of SEQ ID NO:
 7. 23. Thecomposition of claim 21 wherein the C-terminal fragment of lacritinconsists of SEQ ID NO:
 7. 24. The composition of claim 21, wherein thecomposition, further comprises a second anti-bacterial agent.
 25. Thecomposition of claim 24, wherein the second anti-bacterial agent islysozyme.
 26. The composition of claim 25, wherein the weight ratio oflysozyme to the C-terminal fragment of lacritin is from 4:1 to 3:1. 27.A method of treating a corneal infection, said method comprisingselecting a subject having a corneal infection and contacting the corneaof the subject with the composition of claim 21.